Methods for making oligonucleotide probes for the detection and/or quantitation of non-viral organisms

ABSTRACT

A method for preparing probes, as well as several probes for use in qualitative or quantitative hybridization assays are disclosed. The method comprises constructing an oligonucleotide that is sufficiently complementary to hybridize to a region of rRNA selected to be unique to a non-viral organism or group of non-viral organisms sought to be detected, said region of rRNA being selected by comparing one or more variable region rRNA sequences of said non-viral organism or group of non-viral organisms with one or more variable region rRNA sequences from one or more non-viral organisms sought to be distinguished. Hybridization assay probes for Mycobacterium avium, Mycobacterium intracellulare, the Mycobacterium tuberculosis-complex bacteria, Mycoplasma pneumoniae, Legionella, Salmonella, Chlamydia trachomatis, Campylobacter, Proteus mirabilis, Enterococcus, Enterobacter cloacae, E. coli, Pseudomonas group I, Neisseria gonorrhoeae, bacteria, and fungi also are disclosed.

The present application is a divisional of Hogan et al., U.S.application Ser. No. 08/200,866, filed Feb. 22, 1994, now U.S. Pat. No.5,541,308, which is file wrapper continuation of Hogan et al., U.S.application Ser. No. 07/806,929, filed Dec. 11, 1991, now abandoned,which is a file wrapper continuation of Hogan et al., U.S. Ser. No.07/295,208, filed Dec. 9, 1988, now abandoned, which was the Nationalfiling of PCT/US87/03009, filed Nov. 24, 1987, which is acontinuation-in-part of Hogan et al., U.S. application Ser. No.07/083,542, filed Aug. 7, 1987, now abandoned, which is acontinuation-in-part of Hogan et al., U.S. Ser. No. 06/934,244, filedNov. 24, 1986, now abandoned, the entirety of each of these priorapplications including drawings are hereby incorporated by referenceherein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The inventions described and claimed herein relate to probes and assaysbased on the use of genetic material such as RNA. More particularly, theinventions relate to the design and construction of nucleic acid probesand hybridization of such probes to genetic material of target non-viralorganisms in assays for detection and/or quantitation thereof in testsamples of, e.g., sputum, urine, blood and tissue sections, food, soiland water.

2. Introduction

Two single strands of nucleic acid, comprised of nucleotides, mayassociate ("hybridize") to form a double helical structure in which thetwo polynucleotide chains running in opposite directions are heldtogether by hydrogen bonds (a weak form of chemical bond) between pairsof matched, centrally located compounds known as "bases." Generally, inthe double helical structure of nucleic acids, for example, the baseadenine (A) is hydrogen bonded to the base thymine (T) or uracil (U)while the base guanine (G) is hydrogen bonded to the base cytosine (C).At any point along the chain, therefore, one may find the base pairs ATor AU, TA or UA, GC, or CG. One may also find AG and GU base pairs inaddition to the traditional ("canonical") base pairs. Assuming that afirst single strand of nucleic acid is sufficiently complementary to asecond and that the two are brought together under conditions which willpromote their hybridization, double stranded nucleic acid will result.Under appropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids maybe formed.

Broadly, there are two basic nucleic acid hybridization procedures. Inone, known as "in solution" hybridization, both a "probe" nucleic acidsequence and nucleic acid molecules from a test sample are free insolution. In the other method, the sample nucleic acid is usuallyimmobilized on a solid support and the probe sequence is free insolution.

A probe may be a single strand nucleic acid sequence which iscomplementary in some particular degree to the nucleic acid sequencessought to be detected ("target sequences"). It may also be labelled. Abackground description of the use of nucleic acid hybridization as aprocedure for the detection of particular nucleic acid sequences isdescribed in U.S. application Ser. No. 456,729, entitled "Method forDetection, Identification and Quantitation of Non-Viral Organisms,"filed Jan. 10, 1983 (Kohne I), and U.S. application Ser. No. 655,365,entitled "Method For Detecting, Identifying and Quantitating Organismsand Viruses," filed Sep. 4, 1984 (Kohne II), both of which areincorporated by reference, together with all other applications citedherein.

Also described in those applications are methods for determining thepresence of RNA-containing organisms in a samples which might containsuch organisms, comprising the steps of bringing together any nucleicacids from a sample and a probe comprising nucleic acid molecules whichare shorter than the rRNA subunit sequence from which it was derived andwhich are sufficiently complementary to hybridize to the rRNA of one ormore non-viral organisms or groups of non-viral organisms, incubatingthe mixture under specified hybridization conditions, and assaying theresulting mixture for hybridization of the probe and any test samplerRNA. The invention is described to include using a probe which detectsonly rRNA subunit subsequences which are the same or sufficientlysimilar in particular organisms or groups of organisms and is said todetect the presence or absence of any one or more of those particularorganisms in a sample, even in the presence of many non-relatedorganisms.

We have discovered and describe herein a novel method and means fordesigning and constructing DNA probes for use in detecting unique rRNAsequences in an assay for the detection and/or quantitation of any groupof non-viral organisms. Some of the inventive probes herein may be usedto detect and/or quantify a single species or strain of non-viralorganism and others may be used to detect and/or quantify members of anentire genus or desired phylogenetic grouping.

SUMMARY OF THE INVENTION

In a method of probe preparation and use, a single stranddeoxyoligonucleotide of particular sequence and defined length is usedin a hybridization assay to determine the presence or amount of rRNAfrom particular target non-viral organisms to distinguish them fromtheir known closest phylogenetic neighbors. Probe sequences which arespecific, respectively, for 16S rRNA variable subsequences ofMycobacterium avium, Mycobacterium intracellulare and the Mycobacteriumtuberculosis-complex bacteria, and which do not cross react with nucleicacids from each other, or any other bacterial species or respiratoryinfectious agent, under proper stringency, are described and claimed. Aprobe specific to three 23S rRNA variable region subsequences from theMycobacterium tuberculosis-complex bacteria is also described andclaimed, as are rRNA variable region probes useful in hybridizationassays for the genus Mycobacterium (16S 23S rRNA specific), Mycoplasmapneumoniae (5S and 16S rRNA-specific), Chlamydia trachomatis (16S and23S rRNA specific), Enterobacter cloacae (23S rRNA specific),Escherichia coli (16S rRNA specific), Legionella (16S and 23S rRNAspecific), Salmonella (16S and 23S rRNA specific), Enterococci (16S rRNAspecific), Neisseria gonorrhoeae (16s rRNA specific), Campylobacter (16SrRNA specific), Proteus mirabilis (23S rRNA specific), Pseudomonas (23SrRNA specific), fungi (18S and 28S rRNA specific), and bacteria (16S and23S rRNA specific).

In one embodiment of the assay method, a test sample is first subjectedto conditions which release rRNA from any non-viral organisms present inthat sample. rRNA is single stranded and therefore available forhybridization with sufficiently complementary genetic material once soreleased. Contact between a probe, which can be labelled, and the rRNAtarget may be carried out in solution under conditions which promotehybridization between the two strands. The reaction mixture is thenassayed for the presence of hybridized probe. Numerous advantages of thepresent method for the detection of non-viral organisms over prior arttechniques, including accuracy, simplicity, economy and speed willappear more fully from the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is made up of FIGS. 1A and 1B and is a chart of the primarystructure of bacterial 16S rRNA for Escherichia coli, depicting standardreference numbers for bases.

FIG. 2 is made up of FIGS. 2A, 2B, 2C and 2D is a chart of the primarystructure of bacterial 23S rRNA for Escherichia coli, depicting standardreference numbers for bases.

FIG. 3 is made up of FIGS. 3A and 3B and is a chart of the primarystructure of bacterial 5S rRNA for Escherichia coli, depicting standardreference numbers for bases.

FIG. 4 is made up of FIGS. 4A, 4B and 4C an is a chart of the primarystructure for the 18S rRNA for Saccharomyces cerevisiae, depictingstandard reference numbers for bases.

FIG. 5 is made up of FIGS. 5A, 5B, 5C, 5D, 5E and 5F and is a chart ofthe primary structure for the 28S rRNA for Saccharomyces cerevisiae,depicting standard reference numbers for bases.

FIG. 6 is a diagram showing the locations in the 16S rRNA (using E. colireference numbers) which differ between 12 different sets of relatedorganisms. In Example 1, for example 99.7 refers to the difference in16s rRNA between Clostridium botuliniumg and Clostridium subterminale.

FIG. 7 is a diagram showing the locations in the first 1500 bases of 23SrRNA (using E. coli reference numbers) which differ between 12 differentsets of related organisms.

FIG. 8 is a diagram showing the locations in the terminal bases of 23SrRNA (using E. coli reference numbers) which differ between 12 differentsets of related organisms.

FIG. 9 is a schematic representation of the location of probes capableof hybridizing to the 16S rRNA.

FIG. 10 is a schematic representation of the location of probes capableof hybridizing to the first 1500 bases of the 23S rRNA.

FIG. 11 is a schematic representation of the location of probes capableof hybridizing to the terminal bases of 23S rRNA.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms, as used in this disclosure and claims, are definedas:

nucleotide: a subunit of a nucleic acid consisting of a phosphate group,a 5' carbon sugar and a nitrogen containing base. In RNA the 5' carbonsugar is ribose. In DNA, it is a 2-deoxyribose. The term also includesanalogs of such subunits.

nucleotide polymer: at least two nucleotides linked by phosphodiesterbonds.

oligonucleotide: a nucleotide polymer generally about 10 to about 100nucleotides in length, but which may be greater than 100 nucleotides inlength.

nucleic acid probe: a single stranded nucleic acid sequence that willcombine with a complementary single stranded target nucleic acidsequence to form a double-stranded molecule (hybrid). A nucleic acidprobe may be an oligonucleotide or a nucleotide polymer.

hybrid: the complex formed between two single stranded nucleic acidsequences by Watson-Crick base pairings or non-canonical base pairingsbetween the complementary bases.

hybridization: the process by which two complementary strands of nucleicacids combine to form double stranded molecules (hybrids).

complementarity: a property conferred by the base sequence of a singlestrand of DNA or RNA which may form a hybrid or double stranded DNA:DNA,RNA:RNA or DNA:RNA through hydrogen bonding between Watson-Crick basepairs on the respective strands. Adenine (A) usually complements thymine(T) or Uracil (U), while guanine (G) usually complements cytosine (C).

stringency: term used to describe the temperature and solventcomposition existing during hybridization and the subsequent processingsteps. Under high stringency conditions only highly homologous nucleicacid hybrids will form; hybrids without a sufficient degree ofcomplementarity will not form. Accordingly, the stringency of the assayconditions determine the amount of complementarity needed between twonucleic acid strands forming a hybrid. Stringency is chosen to maximizethe difference in stability between the hybrid formed with the targetand the nontarget nucleic acid.

probe specificity: characteristic of a probe which describes its abilityto distinguish between target and non-target sequences. Dependent onsequence and assay conditions. Probe specificity may be absolute (i.e.,probe able to distinguish between target organisms and any nontargetorganisms), or it may be functional (i.e., probe able to distinguishbetween the target organism and any other organism normally present in aparticular sample). Many probe sequences can be used for either broad ornarrow specificity depending on the conditions of use.

variable region: nucleotide polymer which differs by at least one basebetween the target organism and nontarget organisms contained in asample.

conserved region: a region which is not variable.

sequence divergence: process by which nucleotide polymers become lesssimilar during evolution.

sequence convergence: process by which nucleotide polymers become moresimilar during evolution.

bacteria: members of the phylogenetic group eubacteria, which isconsidered one of the three primary kingdoms.

Tm: temperature at which 50% of the probe is converted from thehybridized to the unhybridized form.

thermal stability: Temperature at which 50% of the probe:target hybridsare converted to the single stranded form. Factors which affect thethermal stability can affect probe specificity and therefore, must becontrolled. Whether a probe sequence is useful to detect only a specifictype of organism depends largely on the thermal stability differencebetween probe:target hybrids ("P:T") and probe:nontarget hybrids("P:NT"). In designing probes the Tm P:T minus the Tm P:NT should be aslarge as possible.

In addition to a novel method for selecting probe sequences, we havediscovered that it is possible to create a DNA probe complementary to aparticular rRNA sequence obtained from a single type of targetmicroorganism and to successfully use that probe in a non-cross reactingassay for the detection of that single microorganism, even in thepresence of its known, most closely related taxonomic or phylogeneticneighbors. With the exception of viruses, all prokaryotic organismscontain rRNA molecules including 5S rRNA, 16S rRNA, and a larger rRNAmolecule known as 23S rRNA. Eukaryotes are known to have 5.0S, 5.8S, 18Sand 28S rRNA molecules or analogous structures. (The term "16S like"sometimes is used to refer to the rRNA found in the small ribosomalsubunit, including 18S and 17S rRNA. Likewise the term "23S like" rRNAsometimes is used to refer to the rRNA found in the large ribosomalsubunit. 5.8S rRNA is equivalent to the 5' end of the 23S like rRNA.)These rRNA molecules contain nucleotide sequences which are highlyconserved among all organisms thus far examined. There are known methodswhich allow a significant portion of these rRNA sequences to bedetermined. For example, complementary oligonucleotide primers of about20-30 bases in length can be hybridized to universally conserved regionsin purified rRNA that are specific to the 5S, 16S, or 23S subunits andextended with the enzyme reverse transcriptase. Chemical degradation ordideoxynucleotide-terminated sequencing reactions can be used todetermine the nucleotide sequence of the extended product. Lane, D. J.et al., Proc. Nat'l Acad. Sci, USA 82, 6955-6959 (1985).

In our invention, comparison of one or more sequenced rRNA variableregions from a target organism to one or more rRNA variable regionsequences from a closely related bacterial species is utilized to selecta sequence unique to the rRNA of the target organism. rRNA is preferableto DNA as a probe target because of its relative abundance and stabilityin the cell and because of its patterns of phylogenetic conservation.

Notwithstanding the highly conserved nature of rRNA, we have discoveredthat a number of regions of the rRNA molecule which can vary insequence, can vary even between closely related species and can,therefore, be utilized to distinguish between such organisms.Differences in the rRNA molecule are not distributed randomly across theentire molecule, but rather are clustered into specific regions. Thedegree of conservation also varies, creating a unique pattern ofconservation across the ribosomal RNA subunits. The degree of variationand the distribution thereof, can be analyzed to locate target sites fordiagnostic probes. This method of probe selection may be used to selectmore than one sequence which is unique to the rRNA of a target organism.

We have identified variable regions by comparative analysis of rRNAsequences both published in the literature and sequences which we havedetermined ourselves using procedures known in the art. We use a SunMicrosystems (TM) computer for comparative analysis. The compiler iscapable of manipulating many sequences of data at the same time.Computers of this type and computer programs which may be used oradapted for the purposes herein disclosed are commercially available.

Generally, only a few regions are useful for distinguishing betweenclosely related species of a phylogenetically conserved genus, forexample, the region 400-500 bases from the 5' end of the 16S rRNAmolecule. An analysis of closely related organisms (FIGS. 6, 7 and 8)reveals the specific positions (variable regions) which vary betweenclosely related organisms. These variable regions of rRNA molecules arethe likely candidates for probe design.

FIGS. 5, 6 and 7 display the variations in 16S and 23S rRNA's betweentwo different bacteria with decreasing amounts of similarity betweenthem. Closer analysis of these figures reveals some subtle patternsbetween these closely related organisms. In all cases studied, we haveseen sufficient variation between the target organism and the closestphylogenetic relative found in the same sample to design the probe ofinterest. Moreover, in all cases studied to date, the per centsimilarity between the target organism (or organisms) and the closestphylogenetically related organisms found in the same sample has beenbetween 90% and 99%. Interestingly, there was enough variation evenbetween the rRNA's of Neisseria's gonorrhoeae and meningitidis (SeeExample 21) to design probes--despite the fact that DNA:DNA homologystudies suggested these two species might actually be one and the same.

These figures also show that the differences are distributed across theentire 16S and 23S rRNA's. Many of the differences, nonetheless, clusterinto a few regions. These locations in the rRNA are good candidates forprobe design, with our current assay conditions. We also note that thelocations of these increased variation densities usually are situated inthe same regions of the 16S and 23S rRNA for comparable per centsimilarity values. In this manner, we have observed that certain regionsof the 16S and 23S rRNA are the most likely sites in which significantvariation exists between the target organism and the closestphylogenetic relatives found in a sample. We have disclosed and claimedspecies specific probes which hybridize in these regions of significantvariation between the target organism and the closest phylogeneticrelative found in a sample.

FIGS. 9, 10 and 11 are a schematic representation of the location ofprobes disclosed and claimed herein. Because 16S and 23S RNAs do not, asa rule, contain sequences of duplication longer than about sixnucleotides in length, probes designed by these methods are specific toone or a few positions on the target nucleic acid.

The sequence evolution at each of the variable regions (for example,spanning a minimum of 10 nucleotides) is, for the most part divergent,not convergent. Thus, we can confidently design probes based on a fewrRNA sequences which differ between the target organism and itsphylogenetically closest relatives. Biological and structuralconstraints on the rRNA molecule which maintain homologous primary,secondary and tertiary structure throughout evolution, and theapplication of such constraints to probe diagnostics is the subject ofongoing study. The greater the evolutionary distance between organisms,the greater the number of variable regions which may be used todistinguish the organisms.

Once the variable regions are identified, the sequences are aligned toreveal areas of maximum homology or "match". At this point, thesequences are examined to identify potential probe regions. Twoimportant objectives in designing a probe are to maximize homology tothe target sequence(s) (greater than 90% homology is recommended) and tominimize homology to non-target sequence(s) (less than 90% homology tonontargets is recommended). We have identified the following usefulguidelines for designing probes with desired characteristics.

First, probes should be positioned so as to minimize the stability ofthe probe:nontarget nucleic acid hybrid. This may be accomplished byminimizing the length of perfect complementarity to non-targetorganisms, avoiding G and C rich regions of homology to non-targetsequences, and by positioning the probe to span as many destabalizingmismatches as possible (for example, dG:rU base pairs are lessdestabalizing than some others).

Second, the stability of the probe: target nucleic acid hybrid should bemaximized. This may be accomplished by avoiding long A and T richsequences, by terminating the hybrids with G:C base pairs and bydesigning the probe with an appropriate Tm. The beginning and end pointsof the probe should be chosen so that the length and % G and % C resultin a Tm about 2-10° C. higher than the temperature at which the finalassay will be performed. The importance and effect of various assayconditions will be explained further herein. Third, regions of the rRNAwhich are known to form strong structures inhibitory to hybridizationare less preferred. Finally, probes with extensive self-complementarityshould be avoided.

In some cases, there may be several sequences from a particular regionwhich will yield probes with the desired hybridization characteristics.In other cases, one sequence may be significantly better than anotherwhich differs merely by a single base.

The following chart indicates how, for one embodiment of the inventionuseful in the detection of a nucleic acid in the presence of closelyrelated nucleic acid sequences, unique sequences can be selected. Inthis example, rRNA sequences have been determined for organisms A-E andtheir sequences, represented numerically, are aligned as shown. It isseen that sequence 1 is common to all organisms A-E. Sequences 2-6 arefound only in organisms A, B and C, while sequences 8, 9 and 10 areunique to organism A. Therefore, a probe complementary to sequences 8, 9or 10 would specifically hybridize to organism A.

    ______________________________________                                        Illustrative Pattern of Sequence                                               Relationships Among Related Bacteria                                           Organism  rRNA Sequence                                                     ______________________________________                                        A       1     2      3    4    5    6     7   8   9  10                         B 1 2 3 4 5 6  7 11 12 13                                                     C 1 2 3 4 5 6 14 15 16 17                                                     D 1 18  19  20  21  22  23 24 25 26                                           E 1 18  19  20  21  27  28 29 30 31                                         ______________________________________                                    

In cases where the patterns of variation of a macromolecule are known,for example, rRNA, one can focus on specific regions as likelycandidates for probe design. However, it is not always necessary todetermine the entire nucleic acid sequence in order to obtain a probesequence. Extension from any single oligonucleotide primer can yield upto 300-400 bases of sequence. When a single primer is used to partiallysequence the rRNA of the target organism and organisms closely relatedto the target, an alignment can be made as outlined above. Plainly, if auseful probe sequence is found, it is not necessary to continue rRNAsequencing using other primers. If, on the other hand, no useful probesequence is obtained from sequencing with a first primer, or if highersensitivity is desired, other primers can be used to obtain moresequences. In those cases where patterns of variation for a molecule arenot well understood, more sequence data may be required prior to probedesign.

Thus, in Examples 1-3 below, two 16S-derived primers were used. Thefirst primer did not yield probe sequences which met the criteria listedherein. The second primer yielded probe sequences which were determinedto be useful following characterization and testing for specificity asdescribed. In Example 4, six 23S primers were used prior to locating theprobe sequence set forth.

Once a presumptive unique sequence has been identified, a complementaryDNA oligonucleotide is synthesized. This single stranded oligonucleotidewill serve as the probe in the DNA/rRNA assay hybridization reaction.Defined oligonucleotides may be synthesized by any of several well knownmethods, including automated solid-phase chemical synthesis usingcyanoethylphosphoramidite precursors. Barone, A. D. et al., NucleicAcids Research 12, 4051-4060 (1984). In this method,deoxyoligonucleotides are synthesized on solid polymer supports. Releaseof the oligonucleotide from the support is accomplished by treatmentwith ammonium hydroxide at 60° C. for 16 hours. The solution is driedand the crude product is dissolved in water and separated onpolyacrylamide gels which generally may vary from 10-20% depending uponthe length of the fragment. The major band, which is visualized byultraviolet back lighting, is cut from the gel with a razor blade andextracted with 0.1M ammonium acetate, pH 7.0, at room temperature for8-12 hours. Following centrifugation, the supernatant is filteredthrough a 0.4 micron filter and desalted on a P-10 column (Pharmacia).Other well known methods for construction of synthetic oligonucleotidesmay, of course, be employed.

Current DNA synthesizers can produce large amounts of synthetic DNA.After synthesis, the size of the newly made DNA is examined by gelfiltration and molecules of varying size are generally detected. Some ofthese molecules represent abortive synthesis events which occur duringthe synthesis process. As part of post-synthesis purification, thesynthetic DNA is usually size fractionated and only those moleculeswhich are the proper length are kept. Thus, it is possible to obtain apopulation of synthetic DNA molecules of uniform size.

It has been generally assumed, however, that synthetic DNA is inherentlycomposed of a uniform population of molecules all of the same size andbase sequence, and that the hybridization characteristics of everymolecule in the preparation should be the same. In reality, preparationsof synthetic DNA molecules are heterogeneous and are composed ofsignificant numbers of molecules which, although the same size, are insome way different from each other and have different hybridizationcharacteristics. Even different preparations of the same sequence cansometimes have different hybridization characteristics.

Accordingly, preparations of the same synthetic probe sequence can havedifferent hybridization characteristics. Because of this the specificityof probe molecules from different preparations can be different. Thehybridization characteristics of each preparation should be examined inorder to determine the hybridization conditions which must be used inorder to obtain the desired probe specificity. For example, thesynthetic probe described in Example 4 below has the specificity profiledescribed in Table 14. This data was obtained by using the hybridizationand assay conditions described. A separate preparation of this probewhich has different hybridization characteristics may not have preciselythe same specificity profile when assayed under the conditions presentedin Example 4. Such probe preparations have been made. To obtain thedesired specificity, these probes can be hybridized and assayed underdifferent conditions, including salt concentration and/or temperature.The actual conditions under which the probe is to be used must bedetermined, or matched to extant requirements, for each batch of probesince the art of DNA synthesis is somewhat imperfect.

Following synthesis and purification of a particular oligonucleotidesequence, several procedures may be utilized to determine theacceptability of the final product. The first is polyacrylamide gelelectrophoresis, which is used to determine size. The oligonucleotide islabelled using, for example, ³² P-ATP and T₄ polynucleotide kinase. Thelabelled probe is precipitated in ethanol, centrifuged and the driedpellet resuspended in loading buffer (80% formamide, 20 mM NaOH, 1 mHEDTA, 0.1% bromophenol blue and 0.1% xylene cyanol). The samples areheated for five minutes at 90° C. and loaded onto a denaturingpolyacrylamide gel. Electrophoresis is carried out in TBE buffer (0.1MTris HCl pH 8.3, 0.08M boric acid, 0.002M EDTA) for 1-2 hours at 1,000volts. Following electrophoresis of the oligonucleotide the gel isexposed to X-ray film. The size of the oligonucleotide is then computedfrom the migration of oligonucleotide standards run concurrently.

The sequence of the synthetic oligonucleotide may also be checked bylabelling it at the 5' end with ³² P-ATP and T₄ polynucleotide kinase,subjecting it to standard chemical degradation techniques, Maxam, A. M.and Gilbert, W., Proc. Nat'l. Acad. Sci. USA 74, 560-564 (1980), andanalyzing the products on polyacrylamide gels. Preferably, thenucleotide sequence of the probe is perfectly complementary to thepreviously identified unique rRNA sequence, although it need not be.

The melting profile, including the melting temperature (Tm) of theoligonucleotide/rRNA hybrids should also be determined. One way todetermine Tm is to hybridize a ³² P-labelled oligonucleotide to itscomplementary target nucleic acid at 50° C. in 0.1M phosphate buffer, pH6.8. The hybridization mixture is diluted and passed over a 2 cmhydroxyapatite column at 50° C. The column is washed with 0.1M phosphatebuffer, 0.02% SDS to elute all unhybridized, single-stranded probes. Thecolumn temperature is then dropped 15° C. and increased in 5° C.increments until all of the probe is single-stranded. At eachtemperature, unhybridized probe is eluted and the counts per minute(cpm) in each fraction determined. The number of cpm shown to be boundto the hydroxyapatite divided by the total cpm added to the columnequals the percent hybridization of the probe to the target nucleicacid.

An alternate method for determining thermal stability of a hybrid isoutlined below. An aliquot of hybrid nucleic acid is diluted into 1 mlof either 0.12M phosphate buffer, 0.2% SDS, 1 mM EDTA, 1 mM EGTA or anappropriate hybridization buffer. Heat this 1 ml of solution to 45degrees C. for 5 minutes and place it into a room temperature water bathto cool for 5 minutes. Assay this 1 ml of hybrid containing solutionover a hydroxyapatite column, capturing the hybrid and washing awayunbound probe. If a hybridization solution other than the 0.12Mphosphate buffer is used, then a dilution of the hybridization solutioninto the 0.12M phosphate buffer will be necessary for binding. Keeptaking aliquots of hybrid and diluting into 1 ml of hybridizationsolution or into the standard 0.12M phosphate buffer solution describedabove while raising the heating temperature 5 degrees C. at a time.Continue this until all of the hybrid is dissociated. The point whereone half of the hybrid is converted to the dissociated form isconsidered the Tm. The Tm for a given hybrid will vary depending on thehybridization solution being used because the thermal stability dependsupon the concentration of different salts, detergents, and other soluteswhich effect relative hybrid stability during thermal denaturation.

Because the extent and specificity of hybridization reactions such asthose described herein are affected by a number of factors, manipulationof one or more of those factors will determine the exact sensitivity andspecificity of a particular probe, whether perfectly complementary toits target or not. For example, the base composition of the probe may besignificant because G-C base pairs exhibit greater thermal stability ascompared to A-T base pairs due to additional hydrogen bonding. Thus,hybridization involving complementary nucleic acids of higher G-Ccontent will be stable at higher temperatures.

We have discovered that the length of the target nucleic acid sequenceand, accordingly, the length of the probe sequence can also beimportant. While it is possible for nucleic acids that are not perfectlycomplementary to hybridize, the longest stretch of perfectly homologousbase sequence will normally primarily determine hybrid stability. Whileoligonucleotide probes of different lengths and base composition may beused, oligonucleotide probes preferred in this invention are betweenabout 15 and about 50 bases in length and are at least about 75-100%homologous to the target nucleic acid. For most applications 95-100%homology to the target nucleic acid is preferred.

Ionic strength and incubation temperature should also be taken intoaccount in constructing a probe. It is known that the rate ofhybridization will increase as ionic strength of the reaction mixtureincreases and that the thermal stability of hybrids will increase withincreasing ionic strength. In general, optimal hybridization forsynthetic oligonucleotide probes of about 15-50 bases in length occursapproximately 5° C. below the melting temperature for a given duplex.Incubation at temperatures below the optimum may allow mismatched basesequences to hybridize and can therefore result in reduced specificity.

As to nucleic acid concentration, it is known that the rate ofhybridization is proportional to the concentration of the twointeracting nucleic acid species. Thus, the presence of compounds suchas dextran and dextran sulphate are thought to increase the localconcentration of nucleic acid species and thereby result in an increasedrate of hybridization. Other agents which will result in increased ratesof hybridization are specified in U.S. application Ser. No. 627,795,entitled "Accelerated Nucleic Acid Reassociation Method", filed Jul. 5,1984, Continuation-in-Part thereof, Serial No. (net yet assigned), filedJun. 4, 1987, and U.S. application Ser. No. 816,711, entitled"Accelerated Nucleic Acid Reassociation Method", filed Jan. 7, 1986,both of which are incorporated by reference. On the other hand, chemicalreagents which disrupt hydrogen bonds such as formamide, urea, DMSO, andalcohols will increase the stringency of hybridization.

Selected oligonucleotide probes may be labelled by any of several wellknown methods. Useful labels include radioisotopes as well asnon-radioactive reporting groups. Isotopic labels include ³ H, ³⁵ S, ³²P, ¹²⁵ I, Cobalt and ¹⁴ C. Most methods of isotopic labelling involvethe use of enzymes and include the known methods of nick translation,end labelling, second strand synthesis, and reverse transcription. Whenusing radio-labelled probes, hybridization can be detected byautoradiography, scintillation counting, or gamma counting. Thedetection method selected will depend upon the hybridization conditionsand the particular radioisotope used for labelling.

Non-isotopic materials can also be used for labelling, and may beintroduced by the incorporation of modified nucleotides through the useof enzymes or by chemical modification of the probe, for example, by theuse of non-nucleotide linker groups. Non-isotopic labels includefluorescent molecules, chemiluminescent molecules, enzymes, cofactors,enzyme substrates, haptens or other ligands. We currently prefer to useacridinium esters.

In one embodiment of the DNA/rRNA hybridization assay invention, alabelled probe and bacterial target nucleic acids are reacted insolution. rRNA may be released from bacterial cells by the sonicdisruption method described in Murphy, K. A. et al., U.S. applicationSer. No. 841,860, entitled "Method for Releasing RNA and DNA FromCells", filed Mar. 20, 1986, which is incorporated herein by reference.Other known methods for disrupting cells include the use of enzymes,osmotic shock, chemical treatment, and vortexing with glass beads.Following or concurrent with the release of rRNA, labelled probe may beadded in the presence of accelerating agents and incubated at theoptimal hybridization temperature for a period of time necessary toachieve significant reaction. Following this incubation period,hydroxyapatite may be added to the reaction mixture to separate theprobe/rRNA hybrids from the non-hybridized probe molecules. Thehydroxyapatite pellet is washed, recentrifuged and hybrids detected bymeans according to the label used.

Twenty-one embodiments illustrative of the claimed inventions are setforth below, in which a synthetic probe or probes complementary to aunique rRNA sequence from a target organism, or group of organisms isdetermined, constructed and used in a hybridization assay.

DESCRIPTION OF PARTICULAR EMBODIMENTS

Mycobacterium are acid-fast, alcohol fast, aerobic, non-mobile bacilli.Their lipid content is high and their growth slow. Mycobacterium aviumand Mycobacterium intracellulare are together referred to as M.avium-intracellulare because they are so difficult to differentiate.Recently, the M. avium complex, which includes M. intracellulare, wasshown to be the second most commonly isolated, clinically significantMycobacterium. Good, R. C. et al., J. Infect. Dis. 146, 829-833 (1982).More recent evidence indicates that these organisms are a common causeof opportunistic infection in patients with AIDS (acquired immunedeficiency syndrome). Gill, V. J. et al., J. Clin. Microbio. 22, 543-546(1985). Treatment of such infections in AIDS patients is difficultbecause these organisms are resistant to most antituberculosis drugs.Often a combination of five drugs are used in therapy. The severity ofthese infections also requires rapid diagnosis which, prior to theinvention herein, was not available.

Members of the Mycobacterium tuberculosis complex (Mtb) includeMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanumand Mycobacterium microti. The first three are pathogenic for humanswhile the last is an animal pathogen. These organisms produce slowlydeveloping granulomas on the skin or they may invade internal organs.Tuberculosis of the lungs can be disseminated to other parts of the bodyby the circulatory system, the lymph system, or the intestinal tract.Despite advances in public health and the advent of effectivechemotherapy, Mycobacterial disease, tuberculosis in particular,continues to represent a major world-wide health problem.

The classical method for detecting bacteria in a test sample involvesculturing of the sample in order to expand the number of bacterial cellspresent into observable colony growths which can be identified andenumerated. If desired, the cultures can also be subjected to additionaltesting in order to determine antimicrobial susceptibility. Currently,the most widely used procedures for the detection, isolation andidentification of Mycobacterium species are the acid-fast bacilli (AFB)smear (using either the Ziehl-Neelsen or fluorochrome techniques),culture methods using Lowenstein-Jensen media and Middlebrook media, andbiochemical tests. The AFB relies on the high lipid content ofMycobacterium to retain dye after exposure to acid-alcohol. While theAFB smear test is relatively rapid and simple to perform it does notalways detect Mycobacteria and will not differentiate betweenMycobacterium avium and non-tuberculosis species, between Mycobacteriumintracellulare and non-tuberculosis species, or between Mycobacteriumtuberculosis-complex bacilli and non-tuberculosis species. For accurateidentification of the infecting Mycobacterial species the clinician mustrely on culture results which can require anywhere from 3 to 8 weeks ofgrowth followed by extensive biochemical testing. Other tests have beendeveloped based on the detection of metabolic products fromMycobacterium using carbon-14 labelled substrates. In particular, theBactec (TM) instrument can detect the presence of Mycobacterium within 6to 10 days of the time of innoculation. Gill, V. J., supra. However, thetest does not distinguish Mycobacterium species. It is often importantto make this determination so that particular drugs to which theorganism is susceptible may be prescribed. For traditional culturemethods, this requires an additional 2 to 3 weeks and for the Bactecmethod, an additional 6 to 10 days.

In addition, specific embodiments for Mycoplasma pneumoniae, theMycobacterium, Legionella, Salmonella, Chlamydia trachomatis,Campylobacter, Proteus mirabilis, Enterococcus, Enterobacter cloacae, E.coli, Pseudomonas Group I, bacteria, fungi and Neisseria gonorrhoeae areset forth in the following examples.

As indicated by the below examples, the present invention hassignificant advantages over each of these prior art methods not only inthe enhanced accuracy, specificity and simplicity of the test, but alsoin greatly reducing the time to achieve a diagnosis. The invention makespossible a definitive diagnosis and initiation of effective treatment onthe same day as testing.

EXAMPLE 1

Described below is the preparation of a single stranddeoxyoligonucleotide of unique sequence and defined length which islabelled and used as a probe in a solution hybridization assay to detectthe presence of rRNA from Mycobacterium avium. This unique sequence isspecific for the rRNA of Mycobacterium avium and does not significantlycross-react under the hybridization conditions of this Example, withnucleic acids from any other bacterial species or respiratory infectiousagent, including the closely-related Mycobacterium intracellulare. Thisprobe is able to distinguish the two species, notwithstanding anapproximate 98% rRNA homology between the two species. In this Example,as well as in Examples 2 and 3, sequences for M. avium, M. tuberculosiscomplex, M. intracellulare and related organisms were obtained by usinga specific primer to a highly conserved region in the 16S rRNA. Thesequence of this primer, derived from E. coli rRNA, was 5'-GGC CGT TACCCC ACC TAC TAG CTA AT-3'. 5 nanograms of primer was mixed with 1microgram of each rRNA to be sequenced in the presence of 0.1M KCl and20 mM Tris-HCl pH 8.3 in a final volume of 10 microliters. The reactionswere heated 10 min. at 45° C. and then placed on ice. 2.5 microliters of³⁵ S dATP and 0.5 microliters of reverse transcriptase were added. Thesample was aliquoted into 4 tubes, each tube containing either dideoxyA, G, T, or C. The concentrations of these nucleotides are set forth inLane et al., supra. The samples were incubated at 40° C. for 30 minutes,and were then precipitated in ethanol, centrifuged and the pelletslypholized dry. Pellets were resuspended in 10 microliters formamidedyes (100% formamide, 0.1% bromphenol blue and 0.1% xylene cyanol), andloaded onto 80 cm 8% polyacrylamide gels. The gels were run at 2000volts for 2-4 hours.

Thus, nucleotide sequences for the 16S rRNA of Mycobacterium avium andwhat were considered to be its closest phylogenetic neighbors,Mycobacterium intracellulare and Mycobacterium tuberculosis, weredetermined by the method of Lane, D. J. et al., Proc. Nat. Acad. Sci.USA 82:6955 (1985). In addition to determining the rRNA sequences forthe organisms noted above, a spectrum of clinically significantMycobacterium were also sequenced. These included M. fortuitum, M.scrofulaceum and M. chelonae. Selected members of several genera closelyrelated to Mycobacterium were also sequenced, including Rhodococcusbronchialis, Corynebacterium xerosis and Nocardia asteroides.

Partial rRNA sequences from the above organisms were aligned for maximumnucleotide homology, using commercially available software fromIntelligenetics, Inc., 1975 El Camino Real West, Mountain View, Calif.94040-2216 (IFIND Program). From this alignment, regions of sequenceunique to Mycobacterium avium were determined. The probe was selected sothat it was perfectly complementary to a target nucleic acid sequenceand so that it had a 10% or greater mismatch with the aligned rRNA fromits known closest phylogenetic neighbor. A sequence 38 bases in lengthwas chosen. The number of mismatched bases relative to the Mycobacteriumavium sequence were as follows: Mycobacterium tuberculosis (8);Mycobacterium intracellulare (5); Mycobacterium scrofulaceum (6);Mycobacterium chelonae (12); and Mycobacterium fortuitum (10).

The following CDNA sequence was characterized by the criteria of length,Tm, and sequence analysis as described at pages 7-8 above and wasdetermined to be specific for the rRNA Mycobacterium avium:

ACCGCAAAAGCTTTCCACCAGAAGACATGCGTCTTGAG.

This sequence is complementary to a unique segment found in the 16S rRNAof Mycobacterium avium. The size of the probe is 38 bases. The probe hasa Tm of 74° C. and sequence analysis by the method of Maxam & Gilbert(1980), supra, confirmed that the probe was correctly synthesized. Theprobe is capable of hybridizing to rRNA of M. avium in the regioncorresponding to bases 185-225 of E. coli 16S rRNA.

To demonstrate the reactivity of this sequence for Mycobacterium avium,it was tested as a probe in hybridization reactions under the followingconditions. ³² P-end-labeled oligonucleotide probes were mixed with 1microgram (7×10⁻¹³ moles) of purified rRNA from Mycobacterium avium andreacted in 0.12M PB hybridization buffer (equimolar amounts of Na₂ HPO₄and NaH₂ PO₄), 1 mM EDTA and 0.02% SDS (sodium dodecyl sulfate) at 65°C. for 60 minutes in a final volume of 50 microliters. In separate tubesthe probe was mixed with the hybridization buffer both with and withouttarget present. Following separation on hydroxyapatite as outlined inthe patent applications identified at page 2, supra, the hybrids werequantitated by scintillation counting. These results are presented inTable 1, showing that the probe has a high extent of reaction tohomologous target and very little non-specific binding to thehydroxyapatite.

                  TABLE 1                                                         ______________________________________                                        HYBRIDIZATION OF THE M. AVIUM PROBE                                             TO HOMOLOGOUS TARGET rRNA*                                                                      plus rRNA                                                                              minus rRNA                                       ______________________________________                                        M. avium probe  85-95%   0.5%                                                 ______________________________________                                         ##STR1##                                                                 

Specificity of the probe for M. avium was tested by mixing the ³² Plabeled probe with rRNA released from cells of 29 other species ofmycobacteria by the sonic disruption techniques described in Murphy etal., U.S. application Ser. No. 841,860. 1×10⁸ cells were suspended in0.1 ml 5% SDS and sonicated for 10 minutes at 50-60° C. 1.0 ml ofhybridization buffer (45% sodium diisobutyl sulfosuccinate, 40 mMphosphate buffer pH 6.8 and 1 mM EDTA) was added and the mixtureincubated for 60 minutes at 72° C. Following incubation, 4.0 ml ofhydroxyapatite solution (0.14M sodium phosphate buffer, pH 6.8, 0.02%SDS and 1.0 gram hydroxyapatite per 50 mls solution) was added andincubated for 5 minutes at 72° C. The sample was centrifuged and thesupernatant removed. 4.0 ml wash solution (0.14M sodium phosphate pH6.8) was added and sample was vortexed, centrifuged and the supernatantremoved. The radioactivity bound to the hydroxyapatite was determined byscintillation counting. The results are shown in Table 2 and indicatetha the probe is specific for Mycobacterium avium and does not reactwith any other mycobacterial species, including Mycobacteriumintracellulare.

                  TABLE 2                                                         ______________________________________                                        HYBRIDIZATION OF THE M. AVIUM PROBE                                             TO MYCOBACTERIAL SPECIES                                                        Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Mycobacterium africanum                                                                         25420   1.0                                                   M. asiaticum 25276 1.2                                                        M. avium 25291 87.6                                                           M. bovis 19210 1.2                                                            M. bovis (BCG) 19015 1.0                                                      M. chelonae 14472 0.9                                                         M. flavescens 14474 0.9                                                       M. fortuitum  6841 1.0                                                        M. gastri 15754 1.2                                                           M. gordonae 14470 1.2                                                         M. haemophilum 29548 1.3                                                      M. intracallulare 13950 1.5                                                   M. kansasii 12478 1.2                                                         M. malmoense 29571 1.2                                                        M. marinum  827 1.2                                                           M. nonchromogenicum  1930 1.1                                                 M. phlei 11758 1.3                                                            M. scrofulaceum 19981 1.2                                                     M. shimoidei 27962 2.3                                                        M. simiae 25275 1.2                                                           M. smegmatis e14468.sup.   1.0                                                M. szulgai 23069 1.0                                                          M. terrae 15755 1.2                                                           M. thermoresistibile 19527 1.3                                                M. triviale 23292 1.2                                                         M. tuberculosis (avirulent) 25177 1.4                                         M. tuberculosis (virulent) 27294 1.1                                          M. ulcerans 19423 1.4                                                         M. vaccae 15483 1.2                                                           M. xenopi 19971 1.5                                                         ______________________________________                                    

As shown in Table 3 the probe also did not react with the rRNA from anyof the respiratory pathogens which were also tested by the method justdescribed. Nor did the probe react with any other closely related orphylogenetically more diverse species of bacteria also tested by thatmethod (Table 4).

                  TABLE 3                                                         ______________________________________                                        HYBRIDIZATION OF M. AVIUM PROBE TO                                              RESPIRATORY PATHOGENS                                                           Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Corynebacterium xerosis                                                                          373    0.7                                                   Fusobacterium nucleatum 25586 1.3                                             Haemophilum influenzae 19418 1.3                                              Klebsiella pneumoniae 23357 1.8                                               Legionella pneumophila 33152 0.0                                              Mycoplasma pneumoniae 15531 3.0                                               Neisseria meningitidis 13090 0.0                                              Pseudomonas aeruginosa 25330 0.0                                              Propionibacterium acnes  6919 1.1                                             Streptococcus pneumoniae  6306 0.0                                            Staphylococcus aureus 25923 1.5                                             ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        HYBRIDIZATION OF THE M. AVIUM PROBE TO A                                        PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES                                 Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Acinetobacter calcoaceticus                                                                     33604   0.0                                                   Branhamella catarrahalis 25238 0.6                                            Bacillus subtilis  6051 0.9                                                   Bacteroides fragilis 23745 1.0                                                Campylobacter jejuni 33560 0.4                                                Chromobacterium violaceum 29094 1.7                                           Clostridium perfringens 13124 2.1                                             Deinococcus radiodurans 35073 0.8                                             Derxia gummosa 15994 0.3                                                      Enterobacter aerogenes 13048 0.6                                              Escherichia coli 11775 0.3                                                    Mycobacterium gordonae 14470 1.9                                              Mycoplasma hominis 14027 3.3                                                  Proteus mirabilis 29906 0.0                                                   Psudomonas cepacia 11762 1.0                                                  Rahnella aquatilis 33071 2.1                                                  Rhodospirillum rubrum 11170 0.6                                               Streptococcus mitis  9811 0.9                                                 Vibrio parahaemolyticus 17802 1.2                                             Yersinia enterocolitica  9610 0.4                                           ______________________________________                                    

EXAMPLE 2

After the alignment described in Example 1, the following sequence wascharacterized by the aforementioned criteria of length, Tm and sequenceanalysis and was determined to be specific for Mycobacteriumintracellulare:

ACCGCAAAAGCTTTCCACCTAAAGACATGCGCCTAAAG

The sequence is complementary to a unique segment found in the 16S rRNAof Mycobacterium intracellulare. The size of the probe was 38 bases. Theprobe has a Tm of 75° C. and sequence analysis confirmed that the probewas correctly synthesized. The probe hybridizes to RNA of M.intracellulare in the region corresponding to bases 185-225 of E. coli16S rRNA.

To demonstrate the reactivity of this sequence for the Mycobacteriumintracellulare, the probe was tested in hybridization reactions underthe following conditions. ³² P-end-labelled oligonucleotide probe wasmixed with 1 microgram (7×10⁻¹³ moles) of purified rRNA fromMycobacterium intracellulare and reacted in 0.12M PB (equimolar amountsof Na₂ HPO₄ and NaH² PO₄), 1 mM EDTA and 0.2% SDS (sodium dodecylsulfate) at 65° C. for 60 minutes in a final volume of 50 microliters.In separate tubes the probe was mixed with the hybridization buffer withand without target Mycobacterium intracellulare rRNA present. Followingseparation on hydroxyapatite as outlined previously the hybrids werequantitated by scintillation counting. These results are shown in Table5.

                  TABLE 5                                                         ______________________________________                                        HYBRIDIZATION OF THE M. INTRACELLULARE PROBE                                    TO HOMOLOGOUS TARGET rRNA*/                                                                       plus rRNA                                                                              minus rRNA                                     ______________________________________                                        M. intracellulare probe                                                                         85-95%   0.5%                                               ______________________________________                                         ##STR2##                                                                      -                                                                        

These data shows that the probe has a high extent of reaction to itshomologous target and very little non-specific binding to thehydroxyapatite.

Specificity of the Mycobacterium intracellulare probe was tested bymixing the 32P labelled probe with rRNA released from cells from 29other species of mycobacteria by sonic disruption techniques describedin Murphy et. al. U.S. patent application Ser. No. 841,860. Allhybridization assays were carried out as described in Example 1. Table 6indicates that the probe is specific for Mycobacterium intracellulareand does not react with any other mycobacterial species, includingMycobacterium avium. These results are impressive in view of the 98%rRNA homology to M. avium; 98% homology to M. kansasii; 98% homology toM. asiaticum; and 97% homology to M. tuberculosis.

                  TABLE 6                                                         ______________________________________                                        HYBRIDIZATION OF THE M. INTRACELLULARE PROBE                                    TO MYCOBACTERIAL SPECIES                                                        Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Mycobacterium africanum                                                                         25420   0.9                                                   M. asiaticum 25276 1.1                                                        M. avium 25291 1.3                                                            M. bovis 19210 1.1                                                            M. bovis (BCG) 19015 1.2                                                      M. chelonae 14472 1.0                                                         M. favescens 14474 1.2                                                        M. fortuitum  6841 1.3                                                        M. gastri 15754 1.3                                                           M. gordonae 14470 1.3                                                         M. haemophilum 29548 0.9                                                      M. intracellulare 13950 78.8                                                  M. kansasii 12479 1.1                                                         M. malmoense 29571 1.0                                                        M. marinum  827 0.9                                                           M. nonchromogenicum  1930 1.0                                                 M. phlei 11758 1.1                                                            M. scrofulaceum 19981 1.0                                                     M. shimoidei 27962 1.3                                                        M. simiae 25275 1.1                                                           M. smegmatis e14468.sup.   1.3                                                M. szulgai 23069 1.0                                                          M. terrae 15755 1.4                                                           M. thermoresistibile 19527 1.6                                                M. triviale 23292 1.3                                                         M. tuberculosis (avirulent) 25177 1.2                                         M. tuberculosis (virulent) 27294 1.2                                          M. ulcerans 19423 1.1                                                         M. vaccae 15483 1.0                                                           M. xenopi 19971 1.2                                                         ______________________________________                                    

As shown in Table 7 the probe did not react with the rRNA from any ofthe respiratory pathogens tested in the hybridization assay. Nor did theprobe react with any other closely related or phylogenetically morediverse species of bacteria that were tested (Table 8).

                  TABLE 7                                                         ______________________________________                                        HYBRIDIZATION OF M. INTRACELLULARE PROBE                                        TO RESPIRATORY PATHOGENS                                                        Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Corynebacterium xerosis                                                                          373    2.2                                                   Fusobacterium nucleatum 25586 1.5                                             Haemophilum influenzae 19418 1.3                                              Klebsiella pneumoniae 23357 1.2                                               Legionella pneumophila 33152 1.2                                              Mycoplasma pneumoniae 15531 3.2                                               Neisseria meningitidis 13090 1.1                                              Pseudomonas aeruginosa 25330 1.0                                              Propionibacterium acnes  6919 2.9                                             Streptococcus pneumoniae  6306 1.6                                            Staphylococcus aureus 25923 1.3                                             ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        HYBRIDIZATION OF THE M. INTRACELLULARE PROBE                                    TO A PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES                             Organism           ATCC#   % Probe                                       ______________________________________                                        Acinetobacter calcoaceticus                                                                       33604   1.5                                                 Branhamella catarrhalis 25238 1.8                                             Bacillus subtilis  6051 1.7                                                   Bacteroides fragilis 23745 1.9                                                Campylobacter jejuni 33560 1.9                                                Chromobacterium violaceum 29094 1.4                                           Clostridium perfringens 13124 2.1                                             Deinococcus radiodurans 35073 2.1                                             Derxia gummosa 15994 1.6                                                      Enterobacter aerogenes 13048 1.3                                              Escherichia coli 11775 1.2                                                    Mycobacterium gordonae 14470 2.3                                              Mycoplasma hominis 14027 2.6                                                  Proteus mirabilis 29906 1.2                                                   Pseudomonas cepacia 11762 1.7                                                 Rahnella aquatilis 33071 1.5                                                  Rhodospirillum rubrum 11170 1.4                                               Strptococcus mitis  9811 1.4                                                  Vibrio parahaemolyticus 17802 2.5                                             Yersinia enterocolitica  9610 1.1                                           ______________________________________                                    

EXAMPLE 3

After the alignment described in Example 1, the following sequence wascharacterized by the aforementioned three criteria of size, sequence andTm, and was determined to be specific to the Mtb complex of organisms,Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacteriumbovis, and Mycobacterium microti:

1. TAAAGCGCTTTCCACCACAAGACATGCATCCCGTG.

The sequence is complementary to a unique segment found in the 16S rRNAof the Mtb-complex bacteria. The size of the probe is 35 bases. Theprobe has a Tm of 72° C. and sequence analysis confirmed that the probewas correctly synthesized. It is capable of hybridizing in the regioncorresponding to bases 185-225 of E. coli 16S rRNA.

To demonstrate the reactivity of this sequence for the Mtb complex theprobe was tested in hybridization reactions under the followingconditions. ³² P-end-labelled oligonucleotide probe was mixed with 1microgram (7×10⁻¹³ moles) of purified rRNA from Mycobacteriumtuberculosis and reacted in 0.12M PB hybridization buffer (equimolaramounts of Na₂ HPO₄, and NaH₂ PO₄), 1 mM EDTA and 0.2 SDS (sodiumdodecyl sulfate) at 65° C. for 60 minutes in a final volume of 50microliters. In separate tubes the probe was mixed with thehybridization buffer with and without target rRNA from Mycobacteriumtuberculosis present. Following separation on hydroxyapatite as outlinedpreviously the hybrids were quantitated by scintillation counting. Theresults are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                        HYBRIDIZATION OF Mtb-COMPLEX 16S rRNA DNA PROBE                                 TO HOMOLOGOUS TARGET rRNA*/                                                                      plus rRNA                                                                              minus rRNA                                      ______________________________________                                        Mtb complex probe                                                                              85-95%   0.5%                                                ______________________________________                                         ##STR3##                                                                      -                                                                        

This data shows that the probe has a high extent of reaction tohomologous target and very little non-specific binding to thehydroxyapatite.

Specificity of the probe for the Mtb complex was tested by mixing the ³²P labelled probe with rRNA released from cells of the 4 Mtb complexbacilli and of 25 other mycobacterial species by sonic disruptiontechniques described in Murphy et. al., U.S. patent application Ser. No.841,860. All hybridization assays were carried out as described inExample 1. Table 10 indicates that the probe is specific for organismswithin the Mtb complex and does not react with any other mycobacterialspecies.

                  TABLE 10                                                        ______________________________________                                        HYBRIDIZATION OF Mtb-COMPLEX 16S rRNA DNA PROBE                                 TO MYCOBACTERIAL SPECIES                                                        Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Mycobacterium africanum                                                                         25420   68.1                                                  M. asiaticum 25276 3.4                                                        M. avium 25291 0.9                                                            M. bovis 19210 63.1                                                           M. chelonae 14472 1.1                                                         M. flavescens 14474 0.9                                                       M. fortuitum  6841 1.1                                                        M. gastri 15754 0.8                                                           M. gordonae 14470 1.1                                                         M. haemophilum 29548 0.8                                                      M. intracallulare 13950 1.1                                                   M. kansasii 12479 1.3                                                         M. malmoense 29571 0.9                                                        M. marinum  827 1.1                                                           M. nonchromogenicum  1930 1.1                                                 M. phlei 11758 1.3                                                            M. scrofulaceum 19981 1.1                                                     M. shimoidei 27962 1.0                                                        M. simiae 25275 1.2                                                           M. smegmatis e14468.sup.   0.9                                                M. szulgai 23069 1.1                                                          M. terrae 15755 1.0                                                           M. thermoresistibile 19527 1.0                                                M. triviale 23292 1.2                                                         M. tuberculosis (avirulent) 25177 66.2                                        M. tuberculosis (virulent) 27294 62.4                                         M. ulcerans 19423 0.9                                                         M. vaccae 15483 0.8                                                           M. xenopi 19971 2.6                                                         ______________________________________                                    

As shown in Table 11 the probe did not react with the rRNA from any ofthe respiratory pathogens tested in the hybridization assay. Nor did theprobe react with any other closely related or phylogenetically morediverse species of bacteria that were tested (Table 12).

                  TABLE 11                                                        ______________________________________                                        HYBRIDIZATION OF Mtb-COMPLEX 16S rRNA DNA PROBE                                 TO RESPIRATORY PATHOGENS                                                        Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Corynebacterium xerosis                                                                          373    1.3                                                   Fusobacterium nucleatum 25586 1.0                                             Haemophilum influenzae 19418 1.6                                              Klebsiella pneumoniae 23357 1.2                                               Legionella pneumophila 33152 1.4                                              Mycoplasma pneumoniae 15531 1.1                                               Neisseria meningitidis 13090 1.0                                              Pseudomonas aeruginosa 25330 1.7                                              Propionibacterium acnes  6919 1.2                                             Streptococcus pneumoniae 25923 0.9                                          ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        HYBRIDIZATION OF THE Mtb-COMPLEX 16S rRNA DNA PROBE                             TO A PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES                             Organism           ATCC#   % Probe                                       ______________________________________                                        Acinetobacter calcoaceticus                                                                       33604   1.3                                                 Branhamella catarrhalis 25238 1.5                                             Bacillus subtilis  6051 1.3                                                   Bacteroides fragilis 23745 1.3                                                Campylobacter jejuni 33560 1.1                                                Chromobacterium violaceum 29094 1.0                                           Clostridium perfringens 13124 1.2                                             Deinococcus radiodurans 35073 1.0                                             Derxia gummosa 15994 1.0                                                      Enterobacter aerogenes 13048 1.0                                              Escherichia coli 11775 1.0                                                    Mycobacterium gordonae 14470 1.3                                              Mycoplasma hominis 14027 0.5                                                  Proteus mirabilis 29906 1.0                                                   Pseudomonas cepacia 11762 2.6                                                 Rahnella aquatilis 33071 1.9                                                  Rhodospirillum rubrum 11170 1.0                                               Streptococcus mitis  9811 1.1                                                 Vibrio parahaemolyticus 17802 0.9                                             Yersinia enterocolitica  9610 1.1                                           ______________________________________                                    

Two derivatives of the probe of Example 3 (numbered 2-3 below) were madeand tested:

2. CCGCTAAAGCGCTTTCCACCACAAGACATGCATCCCG

3. ACACCGCTAAAGCGCTTTCCACCACAAGACATGCATC.

All three probes have similar Tms (72°; 73.5°; and 72.3°, respectively)and similar hybridization characteristics.

Hybridization to Mycobacterium tuberculosis complex organisms was 68-75%and non-specific hybridization to hydroxyapatite was less than 2%.Results of hybridization assay tests for these derivatives follow.

                  TABLE 13                                                        ______________________________________                                        HYBRIDIZATION OF PROBE OF EXAMPLES 3 AND 2                                      DERIVATIVES THEREOF                                                           TO MYCOBACTERIAL SPECIES                                                                              Example                                                                   % Probe 1                                                                              % Probe 2                                                                            % Probe 3                                 Organism ATCC#  Bound Bound Bound                                           ______________________________________                                        Mycobacterium 25420   68.1     69.4   70.6                                      africanum                                                                     M. asiaticum 25274 3.4 5.3 1.8                                                M. avium 25291 0.9 1.6 1.4                                                    M. bovis 19210 63.1 75.3 74                                                   M. chelonae 14472 1.1 1.5 1.6                                                 M. flavescens 14474 0.9 2.7 1.4                                               M. fortuitum  6841 1.1 3.6 1.5                                                M. gastri 15754 0.8 3.6 1.7                                                   M. gordonae 14470 1.1 1.6 1.4                                                 M. haemophilum 29548 0.8 3.2 1.7                                              M. intracellulare 13950 1.1 1.6 1.4                                           M. kansasii 12478 1.3 2.1 2.0                                                 M. malmoense 29571 0.9 2.8 1.5                                                M. marinum  827 1.1 2.1 1.5                                                   M. nonchromogenicum  1930 1.1 3.0 1.5                                         M. phlei 11758 1.3 1.3 1.1                                                    M. scrofulaceum 19981 1.1 3.4 1.6                                             M. shimoidei 27962 1.0 2.7 1.6                                                M. simiae 25275 1.2 2.9 1.8                                                   M. smegmatis e14468.sup.   0.9 1.5 1.2                                        M. szulgai 23069 1.1 3.6 1.1                                                  M. terrae 15755 1.0 3.7 2.0                                                   M. thermoresistibile 19527 1.0 1.6 1.3                                        M. triviale 23292 1.2 1.6 2.0                                                 M. tuberculosis 25177 66.2 75 68                                              (avirulent)                                                                   M. tuberculosis 27294 62.4 74 75                                              (virulent)                                                                    M. ulcerans 19423 0.9 1.7 3.0                                                 M. vaccae 15483 0.8 1.4 1.2                                                   M. xenopi 19971 2.6 1.4 1.2                                                 ______________________________________                                    

EXAMPLE 4

The probe specific for the 23S rRNA of the M. tuberculosis complex wasobtained by using a primer which was complementary to a highly conservedregion of 23S rRNA. The sequence of this primer, derived from E. colirRNA, was 5'-AGG AAC CCT TGG GCT TTC GG-3'. Five nanograms of thisprimer was mixed with 1 microgram of rRNA from M. tuberculosis and otherclosely related Mycobacterium and the procedure as described forExamples 1, 2 and 3 was followed. After alignment as described inExample 1, the following sequence was determined to be specific to theMtb complex of organisms, Mycobacterium tuberculosis, Mycobacteriumafricanum, Mycobacterium bovis, and Mycobacterium microti:

TGCCCTACCCACACCCACCACAAGGTGATGT.

The sequence is complementary to a unique segment found in the 23S rRNAof the Mtb-complex bacteria. The oligonucleotide probe was characterizedas previously described by the criteria of length, Tm and sequenceanalysis. The size of the probe is 31 bases. The probe has a Tm of 72.5°C. and sequence analysis confirmed that the probe was correctlysynthesized. It is capable of hybridizing in the region corresponding tobases 1155-1190 of E. coli 23S rRNA.

To demonstrate the reactivity of this sequence for the Mtb complex theprobe was tested in hybridization reactions under the followingconditions. ³² P-end-labelled oligonucleotide probes were mixed with 1microgram (7×10⁻¹³ moles) of purified rRNA from Mycobacteriumtuberculosis and reacted in 0.12M PB hybridization buffer (equimolaramounts of Na₂ HPO₄, and NaH₂ PO₄), 1 mM EDTA and 0.2 SDS (sodiumdodecyl sulfate) at 65° C. for 60 minutes in a final volume of 50microliters. In separate tubes the probe was mixed with thehybridization buffer with and without target rRNA from Mycobacteriumtuberculosis present. Following separation on hydroxyapatite as outlinedpreviously the hybrids were quantitated by scintillation counting. Theresults are shown in Table 14.

                  TABLE 14                                                        ______________________________________                                        HYBRIDIZATION OF THE Mtb-COMPLEX                                                23S rRNA DNA PROBE TO HOMOLOGOUS TARGET rRNA                                                  plus rRNA                                                                              minus rRNA                                         ______________________________________                                        Mtb complex 23S probe                                                                           94%      1.2%                                               ______________________________________                                    

These data show that the probe has a high extent of reaction tohomologous target and very little non-specific binding to thehydroxyapatite.

Specificity of the probe for the Mtb complex was tested by mixing the ³²P labelled probe with rRNA released from cells of the four Mtb complexbacilli and of 25 other mycobacterial species by sonic disruptiontechniques described in Murphy et al., U.S. patent application Ser. No.841,860. All hybridization assays were carried out as described inExample 1. Table 14 indicates that the probe is specific for organismswithin the Mtb complex and does not react with any other mycobacterialspecies.

                  TABLE 15                                                        ______________________________________                                        HYBRIDIZATION OF Mtb-COMPLEX 23S rRNA DNA PROBE                                 TO MYCOBACTERIAL SPECIES                                                        Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Mycobacterium africanum                                                                         25420   33.6                                                  M. asiaticum 25276 1.2                                                        M. avium 25291 1.0                                                            M. bovis 19210 32.0                                                           M. chelonae 14472 1.2                                                         M. flavescens 14474 1.2                                                       M. fortuitum  6841 1.3                                                        M. gastri 15754 1.1                                                           M. gordonae 14470 1.2                                                         M. haemophilum 29548 1.2                                                      M. intracellulare 13950 1.1                                                   M. kansasii 12479 1.3                                                         M. malmoense 29571 1.3                                                        M. marinum  827 1.2                                                           M. nonchromogenicum  1930 1.0                                                 M. phlei 11758 1.0                                                            M. scrofulaceum 19981 1.1                                                     M. shimoidei 27962 1.2                                                        M. simiae 25275 1.3                                                           M. smegmatis e14468.sup.   1.1                                                M. szulgai 23069 1.1                                                          M. terrae 15755 1.0                                                           M. thermoresistibile 19527 1.2                                                M. triviale 23292 1.0                                                         M. tuberculosis (avirulent) 25177 33.7                                        M. tuberculosis (virulent) 27294 38.1                                         M. ulcerans 19423 1.3                                                         M. vaccae 15483 1.0                                                           M. xenopi 19971 1.3                                                         ______________________________________                                    

EXAMPLE 5

Three additional Mycobacterium tuberculosis complex probes, Examples 5-7herein, were identified using two unique primers complementary to 23SrRNA. The first sequence is:

CCATCACCACCCTCCTCCGGAGAGGAAAAGG.

The sequence of this Example 5 was obtained using a 23S primer with thesequence 5'-GGC CAT TAG ATC ACT CC-3'. It was characterized and shown tobe specific for the Mycobacterium tuberculosis complex of organismsincluding Mycobacterium tuberculosis, Mycobacterium africanum andMycobacterium bovis. This sequence, from 23S rRNA, is 31 bases in lengthand has a Tm of 72° C. This probe is capable of hybridizing to RNA ofthe aforementioned organisms in the region corresponding to bases540-575 of E. coli 23S rRNA.

To demonstrate the reactivity and specificity of this probe forMycobacterium tuberculosis complex, it was tested as a probe inhybridization reactions under the following conditions. ³² P-end-labeledoligonucleotide probe was mixed with rRNA released from cells of 30species of mycobacteria by the sonic disruption techniques described inMurphy et al., U.S. patent application Ser. No. 841,860. 3×10⁷ cellswere suspended in 0.1 ml 5% SDS and sonicated for 15 minutes at 50-60°C. One ml of hybridization buffer (45% diisobutyl sulfosuccinate, 40 mMphosphate buffer pH 6.8, 1 mM EDTA, 1 mM EGTA) was added and the mixtureincubated at 72° C. for 2 hours. Following incubation, 4 ml of 2% (w/v)hydroxyapatite, 0.12M sodium phosphate buffer pH6.8, 0.02% SDS, 0.02%sodium azide was added and incubated at 72° C. for 5 minutes. The samplewas centrifuged and the supernatant removed. Four ml wash solution(0.12M sodium phosphate buffer pH6.8, 0.02% SDS, 0.02% sodium azide) wasadded and the sample was vortexed, centrifuged and the supernatantremoved. The radioactivity bound to the hydroxyapatite was determined byscintillation counting. The results are shown in Table 16 and indicatethat the probe is specific for the Mycobacterium tuberculosis complex oforganisms.

                  TABLE 16                                                        ______________________________________                                        HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX                                    PROBE OF EXAMPLE 5 TO MYCOBACTERIAL SPECIES                                     Organism         ATCC #   % Probe Bound                                   ______________________________________                                        Mycobacterium africanum                                                                        25420    18.0                                                  M. asiaticum 25274 2.6                                                        M. avium 25291 3.4                                                            M. bovis 19210 21.7                                                           M. bovis (BCG) 35734 35.3                                                     M. chelonae 14472 3.8                                                         M. flavescens 14474 2.3                                                       M. fortuitum  6841 1.8                                                        M. gastri 15754 2.2                                                           M. gordonae 14470 2.8                                                         M. haemophilum 29548 2.8                                                      M. intracellulare 13950 2.1                                                   M. kansasii 12478 1.6                                                         M. malmoense 29571 2.3                                                        M. marinum  827 2.1                                                           M. nonchromogenicum  1930 2.3                                                 M. phlei 11758 2.1                                                            M. scrofulaceum 19981 2.2                                                     M. shimoidei 27962 1.9                                                        M. simiae 25275 2.2                                                           M. smegmatis e14468.sup.   2.0                                                M. szulgai 23069 2.2                                                          M. terrae 15755 2.2                                                           M. thermoresistibile 19527 2.2                                                M. triviale 23292 2.0                                                         M. tuberculosis (avirulent) 25177 26.4                                        M. tuberculosis (virulent) 27294 36.6                                         M. ulcerans 19423 2.5                                                         M. vaccae 15483 2.4                                                           M. xenopi 19971 2.8                                                         ______________________________________                                    

Table 16 shows that the probe also did not cross react with rRNA fromany of the closely related organisms tested by the method justdescribed.

                  TABLE 17                                                        ______________________________________                                        HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX                                    PROBE OF EXAMPLE 5 TO PHYLOGENETICALLY                                        CLOSELY RELATED ORGANISMS                                                       Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Actinomadura madurae                                                                            19425   2.1                                                   Actinoplanes italicus 10049 3.1                                               Arthrobacter oxidans 14358 2.1                                                Brevibacterium linens e9172 1.9                                               Corynebacterium xerosis  373 2.2                                              Dermatophilus congolensis 14367 2.2                                           Microbacterium lacticum  8180 2.1                                             Nocardia asteroides 19247 2.0                                                 Nocardia brasiliensis 19296 2.2                                               Nocardia otitidis-caviarum 14629 2.0                                          Nocardioposis dassonvillei 23218 4.0                                          Oerskovia turbata 33225 2.2                                                   Oerskovia xanthineolytica 27402 2.0                                           Rhodococcus aichiensis 33611 1.9                                              Rhodococcus aurantiacus 25938 2.0                                             Rhodococcus bronchialis 25592 2.1                                             Rhodococcus chubuensis 33609 2.3                                              Rhodococcus equi  6939 2.4                                                    Rhodococcus obuensis 33610 2.2                                                Rhodococcus sputi 29627 2.3                                                 ______________________________________                                    

EXAMPLE 6

The second Mycobacterium tuberculosis complex probe was obtained using a23S primer with the sequence 5' CCT GAT TGC CGT CCA GGT TGA GGG AAC CTTTGG G-3'. Its sequence is:

CTGTCCCTAAACCCGATTCAGGGTTCGAGGTTAGATGC

This sequence, from 23S rRNA, is 38 bases in length and has a Tm of 75°C. It hybridizes in the region corresponding to bases 2195-2235 of E.coli 23s rRNA.

Like the complex probe in Example 5, this sequence was characterized andshown to be specific for the Mycobacterium tuberculosis complex oforganisms including Mycobacterium tuberculosis, Mycobacterium africanumand Mycobacterium bovis.

To demonstrate the reactivity and specificity of the probe of thisExample 6 to Mycobacterium tuberculosis complex, it was tested as aprobe in hybridization reactions under the following conditionsdescribed for the probe in Example 5. The results are shown in Table 18and indicate that the probe is specific for the Mycobacteriumtuberculosis complex of organisms with the exception of Mycobacteriumthermoresistibile, a rare isolate which is not a human pathogen.

                  TABLE 18                                                        ______________________________________                                        HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX                                    PROBE OF EXAMPLE 6 TO MYCOBACTERIAL SPECIES                                     Organism         ATCC #   % Probe Bound                                   ______________________________________                                        Mycobacterium africanum                                                                        25420    56.0                                                  M. asiaticum 25274 3.1                                                        M. avium 25291 2.6                                                            M. bovis 19210 48.0                                                           M. bovis (BCG) 35734 63.0                                                     M. chelonae 14472 2.8                                                         M. flavescens 14474 2.8                                                       M. fortuitum  6841 3.0                                                        M. gastri 15754 3.2                                                           M. gordonae 14470 3.0                                                         M. haemophilum 29548 3.0                                                      M. intracellulare 13950 3.6                                                   M. kansasii 12478 3.9                                                         M. malmoense 29571 2.9                                                        M. marinum  827 2.9                                                           M. nonchromogenicum  1930 4.8                                                 M. phlei 11758 2.9                                                            M. scrofulaceum 19981 2.6                                                     M. shimoidei 27962 3.6                                                        M. simiae 25275 3.3                                                           M. smegmatis e14468 3.0                                                       M. szulgai 23069 2.8                                                          M. terrae 15755 2.8                                                           M. thermoresistibile 19527 11.7                                               M. triviale 23292 3.2                                                         M. tuberculosis (avirulent) 25177 65.0                                        M. tuberculosis (virulent) 27294 53.0                                         M. ulcerans 19423 2.5                                                         M. vaccae 15483 2.8                                                           M. xenopi 19971 3.3                                                         ______________________________________                                    

Table 19 shows that the probe also did not cross react with RNA from anyof the phylogenetically closely related organisms tested by the methodjust described.

                  TABLE 19                                                        ______________________________________                                        HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX PROBE                              OF EXAMPLE 6 TO PHYLOGENETICALLY CLOSELY                                      RELATED ORGANISMS                                                               Organism         ATCC #   % Probe Bound                                   ______________________________________                                        Actinomadura madurae                                                                           19425    1.3                                                   Actinoplanes italicus 10049 0.6                                               Arthrobacter oxidans 14358 1.1                                                Brevibacterium linens e9172 0.8                                               Corynebacterium xerosis  373 1.0                                              Dermatophilus congolensis 14367 0.6                                           Microbacterium lacticum  8180 1.9                                             Nocardia asteroides 19247 0.9                                                 Nocardia brasiliensis 19296 0.8                                               Nocardia otitidis-caviarum 14629 1.5                                          Nocardioposis dassonvillei 23218 0.5                                          Oerskovia turbata 33225 0.3                                                   Oerskovia xanthineolytica 27402 0.8                                           Rhodococcus aichiensis 33611 1.6                                              Rhodococcus aurantiacus 25938 0.7                                             Rhodococcus bronchialis 25592 1.5                                             Rhodococcus chubuensis 33609 0.8                                              Rhodococcus equi  6939 0.3                                                    Rhodococcus obuensis 33610 0.8                                                Rhodococcus sputi 29627 1.4                                                 ______________________________________                                    

EXAMPLE 7

The following additional Mycobacterium tuberculosis complex probe alsohas been identified using a 23S primer with the same sequence as that ofExample 6, namely, 5'-CCT GAT TGC CGT CCA GGT TGA GGG AAC CTT TGG G-3':

AGGCACTGTCCCTAAACCCGATTCAGGGTTC.

This sequence, from 23S rRNA is 31 bases in length and has a Tm of 71°C. It hybridizes in the region corresponding to bases 2195-2235 of E.coli 23S rRNA. As is the case with the Mycobacterium tuberculosiscomplex probes of Examples 5 and 6 herein, this sequence also wascharacterized and shown to be specific for the Mycobacteriumtuberculosis complex of organisms, including Mycobacterium tuberculosis,Mycobacterium africanum and Mycobacterium bovis.

To demonstrate the reactivity and specificity of this probe forMycobacterium tuberculosis complex, it was tested as a probe inhybridization reactions under the conditions described for the probe ofExample 5. Table 20 shows that the probe is specific for theMycobacterium tuberculosis complex of organisms.

                  TABLE 20                                                        ______________________________________                                        HYBRIDIZATION OF THE MYCOBACTERIUM TUBERCULOSIS                                 COMPLEX PROBE OF EXAMPLE 7 TO MYCOBACTERIAL SPECIES                             Organism         ATCC #   % Probe Bound                                   ______________________________________                                        Mycobacterium africanum                                                                        25420    43.0                                                  M. asiaticum 25274 0.6                                                        M. avium 25291 0.7                                                            M. bovis 19210 43.0                                                           M. bovis (BCG) 35734 46.0                                                     M. chelonae 14472 0.6                                                         M. flavescens 14474 0.6                                                       M. fortuitum  6841 0.5                                                        M. gastri 15754 0.9                                                           M. gordonae 14470 0.7                                                         M. haemophilum 29548 0.6                                                      M. intracellulare 13950 0.6                                                   M. kansasii 12478 0.9                                                         M. malmoense 29571 0.8                                                        M. marinum  827 0.7                                                           M. nonchromogenicum  1930 0.8                                                 M. phlei 11758 0.6                                                            M. scrofulaceum 19981 0.7                                                     M. shimoidei 27962 0.8                                                        M. simiae 25275 0.7                                                           M. smegmatis e14468 0.6                                                       M. szulgai 23069 0.6                                                          M. terrae 15755 0.7                                                           M. thermoresistibile 19527 0.9                                                M. triviale 23292 0.7                                                         M. tuberculosis (avirulent) 25177 40.0                                        M. tuberculosis (virulent) 27294 50.0                                         M. ulcerans 19423 0.7                                                         M. vaccae 15483 0.4                                                           M. xenopi 19971 0.6                                                         ______________________________________                                    

Table 21 shows that the probe also did not cross react with RNA from anyof the closely related organisms tested by the method just described.

                  TABLE 21                                                        ______________________________________                                        HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX PROBE                              OF EXAMPLE 7 TO PHYLOGENETICALLY CLOSELY                                      RELATED ORGANISMS                                                               Organism         ATCC #   % Probe Bound                                   ______________________________________                                        Actinomadura madurae                                                                           19425    1.0                                                   Actinoplanes italicus 10049 0.6                                               Arthrobacter oxidans 14358 0.4                                                Brevibacterium linens e9172 0.8                                               Corynebacterium xerosis  373 0.6                                              Dermatophilus congolensis 14367 0.8                                           Microbacterium lacticum  8180 0.5                                             Nocardia asteroides 19247 0.7                                                 Nocardia brasiliensis 19296 0.5                                               Nocardia otitidis-caviarum 14629 0.6                                          Nocardioposis dassonvillei 23218 0.6                                          Oerskovia turbata 33225 0.8                                                   Oerskovia xanthineolytica 27402 0.6                                           Rhodococcus aichiensis 33611 0.7                                              Rhodococcus aurantiacus 25938 0.7                                             Rhodococcus bronchialis 25592 0.6                                             Rhodococcus chubuensis 33609 0.6                                              Rhodococcus equi  6939 0.6                                                    Rhodococcus obuensis 33610 0.6                                                Rhodococcus sputi 29627 0.9                                                 ______________________________________                                    

Notably, overlapping probes may have identical specificity. Compare, forexample, the probes of Examples 6 and 7:

Ex. 6 CTGTCCCTAAACCCGATTCAGGGTTCGAGGTTAGATGC

Ex. 7 AGGCACTGTCCCTAAACCCGATTCAGGGTTC

There may be several sequences from a particular region which will yieldprobes with the desired hybridization characteristics. In other cases,one probe sequence may be significantly better than another probediffering by a single base. In general, the greater the sequencedifference (% mismatch) between a target and nontarget organism, themore likely one will be able to alter the probe without affecting itsusefulness for a specific application. This phenomenon also wasdemonstrated by the derivative probes in Example 3.

In Example 7, five bases were added to the 5' end of the probe inExample 6, and 12 bases were removed from the 3' end. The two probeshave essentially identical hybridization characteristics.

EXAMPLE 8

The Mycobacterium genus is particularly difficult to distinguish fromNocardia, Corynebacterium and Rhodococcus. These genera have commonantigens, precipitins and G & C counts. Despite the fact that theseorganisms also exhibit 92-94% rRNA homology to the above listedMycobacterium organisms, we have designed probes which detect allmembers of the genus Mycobacterium without cross reacting to the relatedgenera.

In addition to the Mycobacterium species probes already disclosed, fourprobes specific for members of the Mycobacterium genus were identifiedusing one primer complementary to 16S rRNA and one primer complementaryto 23S rRNA. Sequence 1 was obtained using a 16S primer with thesequence 5'-TTA CTA GCG ATT CCG ACT TCA-3'. Sequences 2, 3 and 4 wereobtained using a 23S primer with the sequence 5'-GTG TCG GTT TTG GGTACG-3'. Sequence 1 is capable of hybridizing to RNA of the genusMycobacterium in the region corresponding to bases 1025-1060 of E. coli16S rRNA. Sequences 2-4 hybridize in regions corresponding to thefollowing bases of E. coli 23S rRNA in our numbering system (See FIG.2); 1440-1475; 1515-1555; 1570-1610 in our numbering system.

The following sequences were characterized and shown to be specific forthe genus Mycobacterium:

    1.  CCA TGC ACC ACC TGC ACA CAG GCC ACA AGG                                      - 2.  GGC TTG CCC CAG TAT TAC CAC TGA CTG GTA CGG                             - 3.  CAC CGA ATT CGC CTC AAC CGG CTA TGC GTC ACC TC                          - 4.  GGG GTA CGG CCC GTG TGT GTG CTC GCT AGA GGC                      

Sequence 1, from 16S rRNA, is 30 bases in length and has a Tm of 73°.Sequence 2, from 23S rRNA, is 33 bases in length and has a Tm of 75° C.Sequence 3, from 23S rRNA, is 35 bases in length and has a Tm of 76° C.Sequence 4, from 23S rRNA, is 33 bases in length and has a Tm of 73° C.

To demonstrate the reactivity and specificity of probe 1 for members ofthe genus Mycobacterium, it was tested as a probe in hybridizationreactions under the following conditions. ¹²⁵ I-labeled oligonucleotideprobe was mixed with rRNA released from cells of 30 species ofmycobacteria by the sonic disruption techniques described in Murphy etal., U.S. patent application Ser. No. 841,860. 3×10⁷ cells weresuspended in 0.1 ml 5% SDS and sonicated for 15 minutes at 50-60° C. Oneml of hybridization buffer (45% diisobutyl sulfosuccinate, 40 mM sodiumphosphate pH6.8, 1 mM EDTA, 1 mM EGTA) was added and the mixtureincubated at 72° C. for 2 hours. Following incubation, 2 ml ofseparation solution (containing 2.5 g/l cationic magnetic microspheres,0.17M sodium phosphate buffer pH6.8, 7.5% Triton X-100 (TM), 0.02%sodium azide) was added and incubated at 72° C. for 5 minutes. TheRNA:probe hybrids, bound to the magnetic particles, were collected andthe supernatant removed. One ml wash solution (0.12M sodium phosphatebuffer pH6.8, 14% diisobutyl sulfosuccinate, 5% Triton X-100, 0.02%sodium azide) was added, the particles collected and the supernatantremoved, This step was repeated two times. The radioactivity bound tothe magnetic particles was determined in a gamma counter. The resultsare shown in Table 22 and indicate that the probes hybridize toorganisms in the genus Mycobacterium and that a combination of probeswill detect all members of the genus. Table 23 shows that the probes donot react with other closely related bacteria.

                  TABLE 22                                                        ______________________________________                                        HYBRIDIZATION OF THE MYCOBACTERIUM                                              PROBES 1-4 TO MYCOBACTERIAL SPECIES                                                              % Probe                                                                              % Probe                                                                              % Probe                                                                              % Probe                               Organism ATCC# 1 Bound 2 Bound 3 Bound 4 Bound                              ______________________________________                                        Mycobacterium                                                                          25420   41.5     14.7   17.9   26.7                                    africanum                                                                     M. asiaticum 25274 31.8 20.2 7.9 0.1                                          M. avium 25291 11.7 34.7 10.1 1.6                                             M. bovis 19210 19.4 28.4 44.6 20.9                                            M. bovis 35734 30.0 35.5 17.8 5.6                                             (BCG)                                                                         M. chelonae 14472  8.6 0.7 6.3 0.2                                            M. flavescens 14474 29.8 17.7 2.3 0.9                                         M. fortuitum  6841 34.7 2.2 4.8 0.2                                           M. gastri 15754 27.6 65.1 9.6 22.3                                            M. gordonae 14470 50.7 55.2 3.1 0.4                                           M. haemo- 29548 40.7 60.7 0.4 12.4                                            philum                                                                        M. intra- 13950 38.8 48.3 0.9 5.4                                             cellulare                                                                     M. kansasii 12478 53.4 27.3 24.5 27.8                                         M. malmoense 29571  3.1 38.4 0.8 1.5                                          M. marinum  827 41.7 4.1 4.8 0.1                                              M. nonchrom-  1930 35.0 42.9 0.5 16.4                                         ogenicum                                                                      M. phlei 11758 23.7 0.6 1.8 0.6                                               M. scrofu- 19981 35.1 66.9 0.9 26.4                                           laceum                                                                        M. shimoidei 27962 34.6 1.4 1.3 4.8                                           M. simiae 25275 45.9 44.0 5.3 0.1                                             M. smegmatis e14468 31.3 4.0 5.6 0.1                                          M. szulgai 23069 19.4 22.3 1.5 3.0                                            M. terrae 15755 25.6 21.7 0.4 12.3                                            M. thermo- 19527 20.3 34.5 3.1 17.6                                           resistibile                                                                   M. triviale 23292 37.3 4.6 4.3 0.1                                            M. tuberculosis 25177 38.5 26.3 11.3 23.0                                     (avirulent)                                                                   M. tuberculosis 27294 13.8 12.4 38.4 22.3                                     (virulent)                                                                    M. ulcerans 19423 33.9 28.7 0.4 8.9                                           M. vaccae 15483  8.8 36.2 4.8 3.2                                             M. xenopi 19971 38.4 2.1 3.8 0.2                                            ______________________________________                                    

                  TABLE 23                                                        ______________________________________                                        HYBRIDIZATION OF THE MYCOBACTERIUM PROBES 1-4                                   TO PHYLOGENETICALLY CLOSELY RELATED ORGANISMS                                                    % Probe                                                                              % Probe                                                                              % Probe                                                                              % Probe                               Organism ATCC# 1 Bound 2 Bound 3 Bound 4 Bound                              ______________________________________                                        Actinomadura                                                                           19425   0.2      0.3    0.2    0.1                                     madurae                                                                       Actinoplanes 10049 0.4 0.5 0.3 0.2                                            italicus                                                                      Arthrobacter 14358 0.2 0.4 0.3 0.1                                            oxidans                                                                       Brevibacterium e9172 0.3 0.3 0.3 0.1                                          linens                                                                        Coryne-  373 0.4 0.3 0.3 0.1                                                  bacterium                                                                     xerosis                                                                       Dermatophilus 14367 0.4 0.6 0.3 0.2                                           congolensis                                                                   Micro-  8180 0.2 0.3 0.2 0.1                                                  bacterium                                                                     lacticum                                                                      Nocardia 19247 0.3 0.3 0.4 0.1                                                asteroides                                                                    Nocardia 19296 0.4 0.3 0.6 0.1                                                brasiliensis                                                                  Nocardia 14629 0.4 0.4 1.0 0.3                                                otitidis-                                                                     caviarum                                                                      Nocardioposis 23218 0.3 0.2 0.3 0.1                                           dassonvillei                                                                  Oerskovia 33225 0.2 0.2 0.3 0.1                                               turbata                                                                       Oerskovia 27402 0.2 0.3 0.3 0.1                                               xanthineolytica                                                               Rhodococcus 33611 0.4 0.2 0.1 0.2                                             aichiensis                                                                    Rhodococcus 25938 0.3 0.4 0.3 0.2                                             aurantiacus                                                                   Rhodococcus 25592 0.4 0.3 0.3 0.1                                             bronchialis                                                                   Rhodococcus 33609 0.6 0.4 0.3 0.3                                             chubuensis                                                                    Rhodococcus  6939 0.4 0.4 0.4 0.5                                             equi                                                                          Rhodococcus 33610 0.5 0.5 0.3 0.1                                             obuensis                                                                      Rhodococcus 29627 0.4 0.5 0.4 0.3                                             sputi                                                                       ______________________________________                                    

EXAMPLE 9

Mycoplasmas are small, aerobic bacteria lacking cell walls. Mycoplasmapneumoniae is estimated to cause 8-15 million infections per year. Theinfections may be asymptomatic or range in severity from mild to severebronchitis and pneumonia. The organism is believed to cause about 10% ofpneumonias in the general population and 10-50% of the pneumonias ofmembers of groups in prolonged, close contact such as college studentsand military personnel.

Diagnosis until now has required isolation of the organism in culture ordemonstration of an increase in antibody titer. Culturing of theorganism involves inoculation of respiratory tract specimens onto agaror biphasic media containing bacterial growth inhibitors. Examinationfor growth at 3-4 and 7-10 days is used to establish the presence orabsence of any mycoplasma. Mycoplasma pneumoniae must then be identifiedby hemadsorption (the ability of M. pneumoniae to adhere sheep or guineapig erythrocytes), hemolysis (the ability of M. pneumoniae to producebeta hemolysis of sheep or guinea pig erythrocytes in blood agar),growth inhibition by specific antibodies, or immunofluorescence withspecific antibodies. The present invention has significant advantagesover each of these prior art methods both because of the simplicity ofthe test and because of the greatly reduced time necessary to achieve adiagnosis.

A probe specific for the 5S rRNA of M. pneumoniae was obtained by acomparison of known rRNA sequences. The particular sequences alignedwere from M. pneumoniae, M. gallisepticum and Ureaplasma urealyticum(Rogers, M. J. et al. 1985, Proc. Natl. Acad. Sci. USA, 82 (1160-1164),M. capricolum (Hori, H. et al. 1981, Nucl. Acids Res. 9, 5407-5410) andSpiroplasma sp. (Walker, R. T. et al. 1982 Nucl. Acids Res. 10,6363-6367). The alignments were performed as described above andoutlined at page 6. 5S rRNA can be isolated and sequenced as outlined inRogers et al., or a primer can be made which is complementary to aconserved region in the 5S rRNA and sequencing performed as outlined inExamples 1-4. The conserved region of 5S rRNA is documented in Fox, G.E. and Woese, C. R., 1975, Nature 256: 505-507. The following sequencewas determined to be specific for Mycoplasma pneumoniae:

GCTTGGTGCTTTCCTATTCTCACTGAAACAGCTACATTCGGC.

The sequence is complementary to a unique segment found in the 5S rRNAof Mycoplasma pneumoniae in the region corresponding to bases 65-108 ofE. coli 5S rRNA, and was selected by comparison to 5S rRNA sequencesfrom Mycoplasma gallisepticum, Spiroplasma mirum and Ureaplasmaurealyticum. The oligonucleotide probe was characterized as describedabove. The size of the probe was 42 bases. The probe has a Tm of 71.5°C.

To demonstrate the reactivity of this sequence for Mycoplasmapneumoniae, the probe was tested in hybridization reactions under thefollowing conditions. ³² P-end-labelled oligonucleotide probe was mixedwith 1 microgram (7×10⁻¹³ moles) of purified rRNA from Mycoplasmapneumoniae and reacted in 0.12M PB (equimolar amounts of Na₂ HPO₄ andNaH₂ PO₄), 1 mM EDTA and 0.2% SDS (sodium dodecyl sulfate) at 65° C. for60 minutes in a final volume of 50 microliters. In separate tubes theprobe was mixed with the hybridization buffer with and without targetMycoplasma pneumoniae rRNA present. Following separation onhydroxyapatite as outlined previously the hybrids were quantitated byscintillation counting. These results are shown in Table 24.

                  TABLE 24                                                        ______________________________________                                        HYBRIDIZATION OF THE M. PNEUMONIAE 5S rRNA DNA                                  PROBE TO HOMOLOGOUS TARGET rRNA*/                                                                plus rRNA                                                                              minus rRNA                                      ______________________________________                                        M. pneumoniae 5S probe                                                                         85-95%   0.5%                                                ______________________________________                                         ##STR4##                                                                      -  This data shows that the probe has a high extent of reaction to its        homologous target and very little non-specific binding to the     hydroxyapatite.

Specificity of the M. pneumoniae 5S probe was tested by mixing the 32Plabelled probe with rRNA released from cells from other Mycoplasmaspecies. All hybridization assays were carried out as described inExample 1. Table 25 indicates that the probe is specific for Mycoplasmapneumoniae and does not react with any other Mycoplasma species.

                  TABLE 25                                                        ______________________________________                                        HYBRIDIZATION OF M. PNEUMONIAE PROBE TO                                         OTHER MYCOPLASMA SPECIES                                                    ______________________________________                                        Acholeplasma laidlawii                                                                            14089   3.3                                                 M. buccale 23636 1.7                                                          M. capricolum 23205 2.4                                                       M. columbinsale 33549 1.4                                                     M. faucium 25293 1.4                                                          M. fermentans 15474 1.0                                                       M. gallisepticum 19610 1.8                                                    M. gallopavonis 33551 1.6                                                     M. genitalium 3353c 1.7                                                       M. hominis 14027 1.3                                                          M. orale 23714 1.8                                                            M. pneumoniae 15531 76.0                                                      M. primatum 15497 1.6                                                         M. salivarium 23064 0.6                                                       Spiroplasma mirum  2.3                                                      ______________________________________                                    

As shown in Table 26, the probe did not react with any other closelyrelated or phylogenetically diverse species of bacteria.

                  TABLE 26                                                        ______________________________________                                        HYBRIDIZATION OF M. PNEUMONIAE PROBE TO                                         A PHYLOGENETIC CROSS SECTION OF BACTERIA                                        Organism            ATCC#   % Probe Bound                                 ______________________________________                                        Corynebacterium xerosis                                                                            373    1.4                                                 Haemophilis influenzae 19418 1.4                                              Klebsiella pneumoniae 23357 1.3                                               Legionella pneumophila 33152 1.8                                              Mycobacterium tuberculosis (avir) 25177 1.6                                   Mycoplasma pneumoniae 15531 52                                                Neisseria meningitidis 13077 0.6                                              Propionibacterium acnes  6919 2.0                                             Pseudomonas aeruginosa 25330 1.6                                              Staphylococcus aureus 12598 2.0                                               Streptococcus pneumoniae c6306 1.9                                          ______________________________________                                    

Four additional probe sequences (numbered 2-5 below) specific forMycoplasma pneumoniae were obtained by utilizing four unique primerscomplementary to conserved regions on 16S rRNA. The regions correspond,respectively, to bases 190-230; 450-490; 820-860; and 1255-1290 of E.coli 16s rRNA. Probe sequence #1 was obtained using a primer with thesequence 5'-GGCCGTTACCCCACCTACTAGCTAAT-3'. Probe sequence #2 wasobtained with a primer with the sequence 5'-GTATTACCGCGGCTGCTGGC-3'.Probe sequence #3 was obtained with a primer with the sequence5'-CCGCTTGTGCGGGCCCCCGTCAATTC-3'. Probe sequence #4 was obtained using aprimer with the sequence 5'-CGATTACTAGCGATTCC-3'. Sequencing reactionswere performed as outlined in previous examples. The M. pneumoniaesequences were compared with sequences from Mycoplasma genitalium,Mycoplasma capricolum, Mycoplasma gallisepticum and Spiroplasma mirum.

The following probe sequences were characterized by criteria describedin example one of the parent application and were shown to be specificfor Mycoplasma pneumoniae:

    2.       AATAACGAACCCTTGCAGGTCCTTTCAACTTTGAT                                     - 3.   CAGTCAAACTCTAGCCATTACCTGCTAAAGTCATT                                    - 4.   TACCGAGGGGATCGCCCCGACAGCTAGTAT                                         - 5.   CTTTACAGATTTGCTCACTTTTACAAGCTGGCGAC.                            

Probe #2 is 35 bases in length and has a Tm of 67° C. Probe #3 is 35bases in length and has a Tm of 66° C. Probe #4 is 30 bases in lengthand has a Tm of 69° C. Probe #5 is 35 bases long with a Tm of 66° C.

When the four probes were mixed and used in hybridization assays at 60°C. in the same manner as previous examples, they were found to bespecific for M. pneumoniae. The probes do not cross react with otherrespiratory pathogens or with any organism representing the bacterialphylogenetic tree (Table 28).

                  TABLE 27                                                        ______________________________________                                        HYBRIDIZATION OF MYCOPLASMA PNEUMOMIAE                                          PROBES 2-5 TO MYCOPLASMA SPECIES                                                Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Acholeplasma axanthum                                                                           27378   0.34                                                  Acholeplasma laidlawii 14089 0.30                                             Mycoplasma arginini 23838 0.20                                                Mycoplasma arthritidis 19611 0.49                                             Mycoplasma bovigenitalium 19852 0.18                                          Mycoplasma bovis 25523 0.43                                                   Mycoplasma buccale 23636 0.37                                                 Mycoplasma californicum 33451 0.79                                            Mycoplasma capricolum 23205 0.38                                              Mycoplasma columbinasale 33549 0.54                                           Mycoplasma columborale 29258 0.50                                             Mycoplasma faucium 25293 0.45                                                 Mycoplasma ferinentans 15474 0.27                                             Mycoplasma gallisepticum 19610 0.25                                           Mycoplasma gallopavonis 33551 0.47                                            Mycoplasma genitalium 33530 2.5                                               Mycoplasma hominis 14027 0.52                                                 Mycoplasma hyorhinis 17981 0.46                                               Mycoplasma orale 23714 0.56                                                   Mycoplasma pneumoniae 15531 34.0                                              Mycoplasma primatum 15497 0.71                                                Mycoplasma pulmonis 19612 0.68                                                Mycoplasma salivarium 23064 0.46                                              Spiroplasma citri 29416 0.60                                                  Spiroplasma mirum 29335 0.52                                                ______________________________________                                    

                  TABLE 28                                                        ______________________________________                                        HYBRIDIZATION OF MYCOPLASMA PNEUMONIAE                                          PROBES 2-5 WITH OTHER BACTERIA                                                  Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Actinomyces ieraelii                                                                            10049   1.0                                                   Bacteroides fragilis 23745 1.4                                                Bifidabacterium breve 15700 1.0                                               Bordetella bronchiseptica 10580 0.9                                           Clostridium innocuum 14501 1.0                                                Clostridium pasteurianum  6013 0.9                                            Clostridium perfringens 13124 1.1                                             Clostridium ramosum 25#82 1.0                                                 Corynebacterium xerosis  373 0.8                                              Erysipelothrix rhusiopathiae 19414 1.1                                        Escherichia coli 11775 1.0                                                    Haemophilus influenzae 19418 0.9                                              Klebsiella pneumoniae 15531 1.0                                               Lactobacillus acidophilus  4356 1.4                                           Legionella pneumophila 33154 0.8                                              Listeria monocytogenes 15313 1.2                                              Moraxella osloensis 19976 1.1                                                 Mycobacterium tuberculosis 25177 1.0                                          Neisseria meningitidis 13077 1.0                                              Pasteurella multocida  6529 1.6                                               Peptococcus magnus 14955 0.9                                                  Propionibacterium acnes  6919 1.1                                             Pseudomonas aeruginosa 25330 1.0                                              Staphylococcus aureus 12600 1.0                                               Streptococcus faecalis 19433 1.5                                              Streptococcus mitis  9811 1.0                                                 Streptococcus pneumoniae  6306 1.0                                            Streptococcus pyogenes 19615 1.1                                            ______________________________________                                    

EXAMPLE 10

The genus Legionella contains 22 species which are all potentiallypathogenic for humans. These organisms cause Legionnaires' disease, anacute pneumonia, or Pontiac fever, an acute, non-pneumonic, febrileillness that is not fatal.

Legionella species have also been shown to be responsible for nosocomialpneumonia occuring predominantly among immunocompromised patients.

Legionellosis, which includes Legionnaires' disease and Pontiac fever,is diagnosed on the basis of clinical symptoms, either direct orindirect fluorescence antibody tests, and by culture using a bufferedcharcoal yeast extract (BCYE) agar containing selective antimicrobialagents. There is no single definitive genus test known in the prior art.(See Bergey's Manual of Systematic Bacteriology at page 283, (ed.1984)). The fluorescent antibody tests are not able to identify allspecies of Legionella, but only those few for which antibodies exist.The culture method is not definitively diagnostic for Legionellaspecies.

The oligonucleotide sequences described below, when used as probes in anucleic acid hybridization assay, accurately identify all species ofLegionella. This assay is more sensitive than culture or antibody testsand shortens significantly the time of identification and, thus,diagnosis. The assay, therefore, represents a significant improvementover prior diagnostic methods.

Three probe sequences specific for the genus Legionella were obtained byutilizing three unique primers complementary to conserved regions onboth 16S and 23S rRNA. Sequence 1 was obtained by using a 16S primerwith the sequence 5'-TCT ACG CAT TTC ACC GCT ACA C-3'. Probe sequence 2was obtained with a 23S primer of sequence 5'-CAG TCA GGA GTA TTT AGCCTT-3'. Probe sequence 3 was obtained with a 16S primer of sequence5'GCT CGT TGC GGG ACT TAA CCC ACC AT-3'. Sequencing with these primerswas performed as described for previous examples.

The following three sequences were characterized by the criteriadescribed in Example 1 and were shown to be specific for the genusLegionella. The phylogenetically nearest neighbors Escherichia coli,Pseudomonas aeruginosa, Vibrio parahaemolyticus and Acinetobactercalcoaceticus were used as comparisons with sequences from Legionellaspecies.

    1.    TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC                                   - 2.    GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC                            - 3.    CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG.                      

Sequence 1, from 16S rRNA, is 40 bases in length and has a Tm of 72° C.Sequence 2, from 23S rRNA, is 42 bases in length and has a Tm of 73° C.Sequence 3, from 16S rRNA, is 40 bases in length and has a Tm of 68° C.These sequences are capable of hybridizing to RNA of the genusLegionella in the regions corresponding respectively to, 630-675 of E.coli 16s rRNA; 350-395 of E. coli 23s rRNA; and 975-1020 of E. coli 16srRNA. When mixed together the probes had a combined average Tm of 73° C.Analysis on polyacrylamide gels showed that each probe was the correctlength and sequence analysis demonstrated that each was the correctsequence of bases.

When the three probes were mixed and used in a hybridization assay, theywere found to be specific for the genus Legionella (Tables 29 and 30)and did not cross react with other respiratory pathogens or with anyselected organism from the phylogenetic tree (Tables 31 and 32). Use ofmore than one probe, i.e., a mixture of probes, can result in increasedassay sensitivity and/or in an increase in the number of non-viralorganisms to be detected.

                  TABLE 29                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA                                                     PROBES TO HOMOLOGOUS TARGET rRNA                                                            plus rRNA                                                                              minus rRNA                                           ______________________________________                                        Legionella probe                                                                              80%      1.0%                                                 ______________________________________                                    

                  TABLE 30                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA                                                     PROBES TO LEGIONELLA SPECIES                                                    Organism      ATCC#       % Probes Bound                                  ______________________________________                                        L. anisa      35292       42.0                                                  L. bozemanii 33217 58.0                                                       L. cherrii 35252 69.0                                                         L. dumoffii 33279 57.0                                                        L. erythra CDC#9P1W044C 26.0                                                  L. feeleii 35303 59.0                                                         L. hackeliae 35250 47.0                                                       L. jamestowniensis 35298 20.0                                                 L. jordanis 33623 50.6                                                        L. longbeachae 33484 48.0                                                     L. maceachernii 35300 25.0                                                    L. micdadei 33704 38.0                                                        L. oakridgensis 33761 44.0                                                    L. parisiensis  9060 69.0                                                     L. pneumophila 1*  6736 75.0                                                  L. pneumophila 2  64.0                                                        L. pneumophila 3  73.0                                                        L. pneumophila 4  73.0                                                        L. pneumophila 5  78.0                                                        L. pneumophila 6  75.0                                                        L. pneumophila 7  73.0                                                        L. pneumophila 8  63.0                                                        L. pneumophila 11  75.0                                                       L. rubrilucens 35304 12.0                                                     L. sainthelensi 35248 61.0                                                    L. sainticrucis 35301 24.0                                                    L. spiritensis CDC#MSH9 55.0                                                  L. steigerwaltii  7430 56.0                                                   L. wadsworthii 33877 37.0                                                   ______________________________________                                         *The numbers 1-8 and 11 are serotypes of L. pneumophila.                 

                  TABLE 31                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA PROBES TO                                           RESPIRATORY PATHOGENS                                                           Organisms         ATCC#   % Probe Bound                                   ______________________________________                                        Corynebacterium xerosis                                                                          373    2.1                                                   Haemophilus influenzae 19418 2.3                                              Klebsiella pneumoniae 23357 2.0                                               Mycoplasma pneumoniae 15531 2.3                                               Neisseria meningitidis 13090 2.2                                              Pseudomonas aeruginosa 25330 1.2                                              Propionibacterium acnes  6919 1.6                                             Streptococcus pneumoniae  6306 0.8                                            Staphylococcus aureus 25923 1.6                                             ______________________________________                                    

                  TABLE 32                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA PROBES TO                                           A PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES                               Organisms         ATCC#   % Probe Bound                                   ______________________________________                                        Acinetobacter calcoaceticus                                                                     33604   1.4                                                   Branhamella catarrahalis 25238 2.0                                            Bacillus subtilis  6051 1.9                                                   Bacteroides fragilis 23745 2.2                                                Campylobacter jejuni 33560 1.2                                                Chromobacterium violaceum 29094 1.3                                           Clostridium perfringens 13124 1.9                                             Deinoccoccus radiodurans 35073 1.8                                            Derxia gummosa 15994 2.0                                                      Enterobacter aerogenes 13048 1.4                                              Escherichia coli 11775 1.2                                                    Mycoplasma hominis 14027 1.1                                                  Proteus mirabilis 29906 1.4                                                   Pseudomonas cepacia 11762 1.1                                                 Rahnella aquatilis 33071 1.7                                                  Rhodospirillum rubrum 11170 2.0                                               Streptococcus mitis  9811 2.0                                                 Vibrio parahaemolyticus 17902 2.0                                             Yersinia enterocolitica  9610 1.2                                           ______________________________________                                    

Three additional probe sequences (numbered 4-6) specific for the genusLegionella were obtained by utilizing two primers complementary toconserved regions on 23S rRNA. Sequence 4 was made from a 23S primerwith the sequence 5'-CCT TCT CCC GAA GTT ACG G-3'. Probe sequences 5 and6 were made from a 23S primer of sequence 5'-AAG CCG GTT ATC CCC GGG GTAACT TTT-3". Sequencing with these primers was performed as described forprevious examples.

The following three sequences were characterized by the criteriapreviously described and were shown to be specific for the genusLegionella. The phylogenetically nearest neighbors Escherichia coli,Pseudomonas aeruginosa, Vibrio parahaemolyticus and Actinetobactercalcoaceticus were used for comparisons with sequences from Legionellaspecies.

    4.     GCG GTA CGG TTC TCT ATA AGT TAT GGC TAG C                                 - 5.  GTA CCG AGG GTA CCT TTG TGC T                                           - 6.  CAC TCT TGG TAC GAT GTC CGA C                                    

Probe 4, complementary to 23S rRNA in the region corresponding to bases1585-1620 of E. coli 23s rRNA, is 31 bases long and has a Tm of 67° C.Probe 5, complementary to 23S rRNA in the region corresponding to bases2280-2330 of E. coli 23s rRNA, is 22 bases long and has a Tm of 66° C.Probe 6, complementary to 23S rRNA in the same region as Probe 5, is 22bases long and has a Tm of 63° C.

When the three probes were mixed with probe 3 above and used in ahybridization assay as described for probes 1-3, they were found to bespecific for the genus Legionella (Table 33) and did not cross reactwith other respiratory pathogens or with any selected organism from thephylogenetic tree (Tables 34 and 35). Using more than one probe, i.e., amixture of probes, can improve assay sensitivity and/or increase thenumber of non-viral organisms detected.

                  TABLE 33                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA PROBES TO                                           LEGIONELLA SPECIES                                                              Organism      ATCC#        % Probes Bound                                 ______________________________________                                        L. anisa      35292        29.6                                                 L. bozeamanii 33217 35.5                                                      L. cherrii 35252 29.2                                                         L. dumoffii 33279 26.0                                                        L. erythra 35303 32.0                                                         L. feelii CDC#9P1WO44C 32.0                                                   L. hackeliae 35250 39.0                                                       L. jamestowniensis 35298 31.2                                                 L. jordanis 33623 25.7                                                        L. longbeachae 33484 27.6                                                     L. maceahernii 35300 39.3                                                     L. micdadei 33204 31.0                                                        L. oakridgensis 33761 24.4                                                    L. parisiensi 35299 31.2                                                      L. pneumophila 1* 33153 40.0                                                  L. pneumophila 2 33154 38.5                                                   L. pneumophila 3 33155 44.6                                                   L. pneumophila 4 33156 48.6                                                   L. pneumophila 5 33216 32.0                                                   L. pneumophila 6 33215 43.0                                                   L. pneumophila 7 33823 29.5                                                   L. pneumophila 8 35096 37.6                                                   L. pneumophila 11 43130 44.5                                                  L. rubrilucens 35304 30.1                                                     L. sainthelensis 35248 27.0                                                   L. sainticrucis 35301 22.0                                                    L. spiritensis CDC#MSH9 40.5                                                  L. steigerwaltii 35302 31.7                                                   L. wadsworthii 33877 30.0                                                   ______________________________________                                         *The numbers 1-8 and 11 are serotypes of L. pneumophila.                 

                  TABLE 34                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA PROBES TO                                           RESPIRATORY PATHOGENS                                                           Organisms         ATCC#   % Probe Bound                                   ______________________________________                                        Corynebacterium xerosis                                                                          373    0.13                                                  Haemophilum influenzae 19418 0.12                                             Klebsiella pneumoniae 23357 0.13                                              Neisseria meningitidis 13090 0.14                                             Pseudomonas aeruginosa 25330 0.13                                             Propionibacterium acnes  6919 0.11                                            Streptococcus pneumoniae  6306 0.08                                           Staphylococcus aureus 25923 0.15                                            ______________________________________                                    

                  TABLE 35                                                        ______________________________________                                        HYBRIDIZATION OF LEGIONELLA PROBES TO                                           A PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES                               Organisms         ATCC#   % Probe Bound                                   ______________________________________                                        Acinetobacter calcoaceticus                                                                     33604   0.12                                                  Branhamella catarrahalis 25238 0.13                                           Bacillus subtilis  6051 0.09                                                  Bacteroides fragilis 23745 0.12                                               Campylobacter jejuni 33560 0.06                                               Chromobacterium violaceum 29094 0.33                                          Clostridium perfringens 13124 0.07                                            Deinoccoccus radiodurans 35073 0.11                                           Derxia gummosa 15994 0.15                                                     Enterobacter aerogenes 13048 0.26                                             Escherichia coli 11775 0.09                                                   Mycoplasma hominis 14027 0.O9                                                 Proteus mirabilis 29906 0.09                                                  Pseudomonas cepacia 17762 0.20                                                Rahnella aquatilis 33071 0.15                                                 Rhodospirillum rubrum 11170 0.13                                              Streptococcus mitis  9811 0.07                                                Vibrio parahaemolyticus 17802 0.11                                            Yersinia enterocolitica  9610 0.19                                          ______________________________________                                    

EXAMPLE 11

Chlamydia are gram-negative, non-motile, obligate intracellularbacteria. The species C. trachomatis is associated with endemic trachoma(the most common preventable form of blindness), inclusionconjunctivitis and lymphogranuloma venereum (LGV). It is a major causeof nongonococcal urethritis in men and may cause cervicitis and acutesalpingitis in women. Eye disease or chlamydial pneumonia may develop innewborns passing through the infected birth canal.

There are several methods known in the art for identification of C.trachomatis in the urogenital tract, for example, by directimmunofluorescent staining or enzyme immunoassay of clinical specimens.The method of choice, however, remains culture of the organism incycloheximide treated McCoy cells. Cell culture is followed bymorphological or fluorescent antibody staining for confirmation of theorganism's identity.

The inventive oligonucleotide sequences described below, when used asprobes in nucleic acid hybridization assay, accurately identifyChlamydia trachomatis isolates. This assay test is equal in sensitivityto culture or antibody tests and, in the case of culture, significantlyshortens the time to identification, and thus, diagnosis.

The use of probes to identify and distinguish between members of thespecies is novel and inventive. Indeed, Kingsbury, D. T., and E. Weiss,1968 J. Bacteriol. 96: 1421-23 (1968); Moulder, J. W., ASM News, Vol.50,No.8, (1984) report a 10% DNA homology between C. trachomatis and C.psittaci. Moreover, these reports show that different C. trachomatisstrains differ in DNA homology. Weisberg, W. G. et. al, J. Bacteriol.167:570-574 (1986) published the 16S rRNA sequences of C. psittaci andnoted that C. trachomatis and C. psittaci share a greater than 95% rRNAhomology. From these reports, it may be inferred that it would bedifficult to invent (1) probes capable of hybridizing to all strains ofC. trachomatis; and (2) probes capable of distinguishing between C.trachomatis and C. psittaci. The following probes accomplish bothobjectives.

Ten probe sequences specific for Chlamydia trachomatis were made usingseven unique primers complementary to conserved regions of both 16S and23S rRNA. Probe sequence 1 was obtained from a 16S primer of sequence5'-TCT ACG CAT TTC ACC GCT ACA C-3'. Probe sequence 2 was obtained witha 16S primer of sequence 5'-CCG CTT GTG CGG GCC CCC GTC AAT TC-3'.Sequences 3 and 4 were obtained using a 16S primer with the sequence5'-GGC CGT TAC CCC ACC TAC TAG CTA AT-3'. Probe sequences 5 and 6 wereobtained with a 23S primer of sequence 5'-CTT TCC CTC ACG GTA-3'. Probesequences 7 and 8 were obtained with a 23S primer of sequence 5'-CCT TCTCCC GAA GTT ACG G-3'. Probe sequence 9 was obtained with a 23S primer ofsequence 5'-TCG GAA CTT ACC CGA CAA GGA ATT TC-3'. Probe sequence 10 wasobtained with a primer of sequence 5'-CTA CTT TCC TGC GTC A-3'.

The following ten sequences were characterized using the criteriadescribed in Example 1 and were shown to be specific for the rRNA ofChlamydia trachomatis. The phylogenetically nearest neighbor Chlamydiapsittaci was used for comparison with Chlamydia trachomatis sequence.

     1. CCG ACT CGG GGT TGA GCC CAT CTT TGA CAA                                      -  2.   TTA CGT CCG ACA CGG ATG GGG TTG AGA CCA TC                            -  3.   CCG CCA CTA AAC AAT CGT CGA AAC AAT TGC TCC GTT                         CGA                                                                         -  4.   CGT TAC TCG GAT GCC CAA ATA TCG CCA CAT TCG                           -  5.   CAT CCA TCT TTC CAG ATG TGT TCA ACT AGG AGT CCT                         GAT CC                                                                      -  6.   GAG GTC GGT CTT TCT CTC CTT TCG TCT ACG                               -  7.    CCG TTC TCA TCG CTC TAC GGA CTC TTC CAA TCG                          -  8.   CGA AGA TTC CCC TTG ATC GCG ACC TGA TCT                               -  9.   CCG GGG CTC CTA TCG TTC CAT AGT CAC CCT AAA AG                        - 10.   TAC CGC GTG TCT TAT CGA CAC ACC CGC G                          

Sequence 1, from 16S rRNA, is 30 bases in length and has a Tm of 66° C.Sequence 2, from 16S rRNA, is 32 bases in length and has a Tm of 67° C.Sequence 3, from 16S rRNA, is 39 bases in length and has a Tm of 70° C.Sequence 4, from 16S rRNA, is 33 bases in length and has a Tm of 69° C.Sequence 5, from 23S rRNA, is 41 bases in length and has a Tm of 71° C.Sequence 6, from 23S rRNA, is 30 bases in length and has a Tm of 72° C.Sequence 7, from 23S rRNA, is 33 bases in length and has a Tm of 72 C.Sequence 8, from 23S rRNA, is 30 bases in length and has a Tm of 71° C.Sequence 9, from 23S rRNA is 35 bases in length and has a Tm of 74° C.Sequence 10 is 28 bases in length and has a Tm of 72° C.

The reactivity and specificity of the probes was tested hybridizationassays. ³² P-end-labeled oligonucleotide probes 1 and 2 were mixed withpurified RNA or RNA released from at least 10⁷ organisms in 0.55 ml of41% diisobutyl sulfosuccinate, 3% sodium dodecyl sulfate, 0.03M sodiumphosphate pH 6.8, 1 mM EDTA, 1 mM EGTA at 60° C. (probe 1) or 64° C.(probe 2) for 1 hour. Hybrids were bound to hydroxyapatite as describedin previous examples and the amount of radioactivity bound wasdetermined by scintillation counting. Table 36 shows that probes 1 and 2hybridize well to all serotypes of C. trachomatis tested. Probe 1 doesnot react with any strain of C. psittaci tested and probe 2 does notreact with two of the strains. Probe 2 does react with the ovinepolyarthritis strain of C. psittaci, an organism which is not known toinfect humans. Table 37 demonstrates the reactivity and specificity ofprobes 3-9 when ¹²⁵ I-labeled and used as a mix. In this case, thehybrids were bound to cationic magnetic particles as described in Arnoldet al., U.S. patent application Ser. No. 020,866 filed Mar. 2, 1987.These probes hybridize well to all strains of C. trachomatis tested andnot to any strains of C. psittaci. Probes 3-9 were further testedagainst a panel of organisms commonly found in the urogenital tract(Table 38) and a phylogenetic cross section of organisms (Table 39). Inall cases, the probes were shown to be specific. Probe 10 is 25%non-homologous to C. psittaci and also should be specific for C.trachomatis.

                  TABLE 36                                                        ______________________________________                                        HIYBRIDIZATION OF CHLAMYDIA TRACHOMATIS PROBES 1 AND                            2 TO CHLAMYDIA RNA                                                                                          % Probe Bound                                 Organism          ATCC#     Probe 1 Probe 2                                   ______________________________________                                        Chlamydia trachomatis serotype C                                                                VR578     22      39                                          Chlamydia trachomatis serotype E VR348B 27 48                                 Chlamydia trachomatis serotype G VR878 20 44                                  Chlamydia trachomatis serotype I VR880 20 42                                  Chlamydia trachomatis serotype K VR887 28 45                                  Chlamydia psittaci guinea pig VR813 1.2 1.4                                   conjunctivitis strain                                                         Chlamydia psittaci ovine VR656 1.0 3.0                                        abortion strain                                                               Chlamydia psittaci ovine poly- VR619 1.1 35.3                                 arthritis strain                                                            ______________________________________                                    

                  TABLE 37                                                        ______________________________________                                        HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS                                          PROBES 3-9 WITH CHLAMYDIA rRNA                                                                               Ratio Counts                                   Organism Serovar ATCC# Bound*                                               ______________________________________                                        C. trachomatis                                                                             A                 689                                              C. trachomatis B  560                                                         C. trachomatis Ba  1066                                                       C. trachomatis C VR548 962                                                    C. trachomatis D  1192                                                        C. trachomatis E VR348 1022                                                   C. trachomatis F  391                                                         C. trachomatis G VR878 874                                                    C. trachomatis H  954                                                         C. trachomatis I VR880 943                                                    C. trachomatis J  482                                                         C. trachomatis K VR887 999                                                    C. trachomatis L1  638                                                        C. trachomatis L2  501                                                        C. trachomatis L3 VR903 821                                                   C. psittaci  VR125 1.6                                                        C. psittaci  VR629 0.9                                                        C. psittaci  VR656 1.3                                                        C. psittaci  VR813 1.2                                                      ______________________________________                                         ##STR5##                                                                      -                                                                        

                  TABLE 38                                                        ______________________________________                                        HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS PROBES 3-9                               TO ORGANISMS FOUND IN THE UROGENITAL TRACT.                                                                 Ratio Counts                                    Organism ATCC# Bound*                                                       ______________________________________                                        Achromobacter xylosoxidans                                                                        27061   1.9                                                 Acinetobacter lwoffii 15309 1.2                                               Branhamella catarrhalis 25238 1.2                                             Candida albicans 18804 2.4                                                    Flavobacterium meningosepticum 13253 1.1                                      Gardnerella vaginalis 14018 1.3                                               Lactobacillus acidophilus  4356 0.8                                           Listeria monocytogenes 15313 0.7                                              Mycobacterium smegmatis 14468 1.1                                             Moraxella osloensis 19976 1.3                                                 Neisseria gonorrhoeae 19424 2.3                                               Pasteurella multocida  6529 1.0                                               Peptostreptococcus anaerobius 27337 1.2                                       Streptococcus agalactiae 13813 4.0                                            Streptococcus faecalis 19433 2.6                                            ______________________________________                                         ##STR6##                                                                      -                                                                        

                  TABLE 39                                                        ______________________________________                                        HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS PROBES 3-9                               TO PHYLOGENETICALLY DIVERSE ORGANISMS.                                                                    Ratio Counts                                      Organism ATCC# Bound*                                                       ______________________________________                                        Bacillus subtilis  6051   2.2                                                   Bacteroides fragilis 23745 1.6                                                Campylobacter jejuni 33560 1.4                                                Chromabacterium violaceum 29094 1.4                                           Deinococcus radiodurans 35073 1.8                                             Derxia gummosa 15994 1.3                                                      Enterobacter aerogenes 13048 1.9                                              Escherichia coli 11775 1.9                                                    Mycoplasma hominis 14027 1.3                                                  Pseudomonas cepacia 17762 2.2                                                 Proteus mirabilis 29906 2.2                                                   Rahnella aquatilis 33071 1.9                                                  Rhodospirillum rubrum 11170 1.9                                               Vibrio parahamolyticus 17802 2.0                                              Yersinia enterocolitica  9610 2.5                                           ______________________________________                                         ##STR7##                                                                      -                                                                        

EXAMPLE 12

Campylobacters are motile, microaerophilic, gram negative curved rods.The genus is quite diverse and distinct from other genera. Although thegenus is well defined, some revision is occurring at the species level(Romaniuk, P. J. et al., J. Bacteriol. 169:2137-2141 (1987). ThreeCampylobacter species, Campylobacter jejuni, C. coli and C. laridis,cause enteritis in humans. The disease includes diarrhea, fever, nausea,abdominal pain and in some cases, vomiting. These organisms cause anestimated 2 million infections per year in the United States (estimatebased on the number of Salmonella and Shigella induced cases ofdiarrheal disease). Other members of the genus cause septicemias inhumans and abortion and infertility in sheep and cattle.

Diagnosis of Campylobacter enteritis is currently dependent upon growthand isolation of the organism in culture, followed by a number ofbiochemical tests. Optimum growth of campylobacters requires specialconditions such as low oxygen tension and high temperature (42° C.). Nosingle set of conditions is recommended for isolation of allCampylobacter species.

The oligonucleotide sequences listed below, when used in a hybridizationassay, hybridize to the 16S rRNA of the Campylobacter species ofinterest. The present invention has significant advantages over theprior art methods of detection of Campylobacter because one probe candetect all Campylobacters of interest; the other two probes detect theenteric Campylobacters and one can detect human isolates ofCampylobacter. In addition, the probes have advantages over the priorart in terms of ease of the assay and greatly reduced time toidentification and therefore, diagnosis.

The four probes which hybridize to the 16S rRNA of Campylobacter speciesof interest were constructed using three unique primers complementary to16S rRNA. Sequences 1 and 2 were made using a 16S primer with thesequences 5'-GTA TTA CCG CGG CTG CTG GCA C-3'. Sequence 3 was made usinga 16S primer with the sequence 5'-CCG CTT GTG CGG GCC CCC GTC AAT TC-3'.Sequence 4 was made with a 16S primer with the sequence 5'-GCT CGT TGCGGG ACT TAA CCC AAC AT-3'.

The following sequences were characterized and shown to hybridize toCampylobacter jejuni, C. coli and C. laridis. The phylogeneticallynearest neighbors Vibrio parahaemolyticus and Wollinella succinogeneswere used for comparison with the campylobacter sequences.

    1.       CGC TCC GAA AAG TGT CAT CCT CC                                          - 2.   CCT TAG GTA CCG TCA GAA TTC TTC CC                                     - 3.   GCC TTC GCA ATG GGT ATT CTT GGT G                                      - 4.   GGT TCT TAG GAT ATC AAG CCC AGG                                 

Sequence 1, from 16S rRNA, is 23 bases in length and has a Tm of 65° C.Sequence ², from 16S rRNA, is 26 bases in length and has a Tm of 64° C.Sequence 3, from 16S rRNA, is 25 bases in length and has a Tm of 66° C.Sequence 4, from 16S rRNA, is 24 bases in length and has a Tm of 61° C.Sequence 1 is capable of hybridizing in the region corresponding tobases 405-428 of E. coli 16s rRNA; Sequence 2 is capable of hybridizingin the region corresponding to bases 440-475 of E. coli 16s rRNA;Sequence 3 is capable of hybridizing in the region corresponding tobases 705-735 of E. coli 16s rRNA; Sequence 4 is capable of hybridizingin the region corresponding to bases 980-1010 of E. coli 16s rRNA.

The reactivity and specificity of the probes for campylobacter wastested in hybridization assays. ³² P-end-labeled oligonucleotide probeswere mixed with purified RNA or RNA released from cells in 0.1% sodiumdodecyl sulfate. 0.5 ml of hybridization solution (41% diisobutylsulfosuccinate, 30 mM sodium phosphate, pH 6.8, 0.7% sodium dodecylsulfate, 1 mM EDTA, 1 mM EGTA) was added and the mixture incubated at60° C. for 1 to 1.5 hour. Following incubation, 2 to 2.5 ml ofseparation solution (2% hydroxyapatite, 0.12M sodium phosphate, pH6.8,0.02% sodium dodecyl sulfate) was added and the mixture incubated at 60°C. for five minutes. The sample was centrifuged and the supernatantremoved. 2.5 ml of wash solution (0.12M sodium phosphate, pH6.8, 0.02%sodium dodecyl sulfate) was added and the sample mixed, centrifuged andthe supernatant removed. The radioactivity bound to the hydroxyapatitewas determined by scintillation counting.

Table 40 indicates that the probes hybridize well to the Campylobacterspecies of interest, C. jejuni, C. coli, and C. laridis. Probe 1 detectsall of the Campylobacter species tested, probes 2 and 4 detect only theenteric campylobacters, and probe 3 detects all of the Campylobacterspecies except C. sputorum, an organism isolated from cattle. Thus allof the probes are useful for identifying campylobacter in stool samples.The choice of which probe to use for other applications would dependupon the level of specificity required (i.e., enteric campylobacters, orall Campylobacter species).

                  TABLE 40                                                        ______________________________________                                        HYBRIDIZATION OF CAMPYLOBACTER PROBES 1-4                                       TO CAMPYLOBACTER SPECIES.                                                                     % Probe Bound (*)                                           Organism     ATCC#    1     2      3    4                                     ______________________________________                                        Campylobacter coli                                                                         33559    64    70     52   49                                      C. fetus 27374 68 0.1 66 0.5                                                  subsp. fetus                                                                  C. fetus 19438 66 0.7 54 1.2                                                  subsp. venerealis                                                             C. jejuni 33560 63 76 51 56                                                   C. laridis 35221 74 73 64 52                                                  C. sputorum 33562 71 3.0 2.5 0                                                subsp. bubulus                                                              ______________________________________                                         % Probe Bound = cpm bound to hybroxyapatitecpm bound when no RNA              present/total cpm used in the assay                                      

Table 41 shows that the probes do not hybridize to closely relatedorganisms or organisms found in the gastrointestinal tract.

                  TABLE 41                                                        ______________________________________                                        HYBRIDIZATION OF CAMPYLOBACTER PROBES 1-4                                       TO CLOSELY RELATED ORGANISMS AND ORGANISMS                                    FOUND IN THE GASTRO-INTESTINAL TRACT.                                                            % Probe Bound (*)                                        Organism        ATCC#    1     2     3   4                                    ______________________________________                                        Bacteroides fragilis                                                                          25285    0     0.2   0.7 0                                      Escherichia coli 11775 1.3 0.5 0.5 0                                          Salmonella typhimurium 14028 0 0 0.3 0                                        Shigella boydii 29929 0 0.2 0.5 0                                             Shigella dysenteriae 13313 0 0.7 0.2 0                                        Shigella flexneri 29903 0 0 0.5 0                                             Shigella sonnei 29930 0 0 0.1 0                                               Vibrio parahaemolyticus 17802 0 1.9 0.1 0                                     Wollinella succinogenes 29543 0.4 2.1 2.2 0                                   Yersinia pseudotuberculosis 29833 0.6 0.2 1.7 0.3                           ______________________________________                                         (*) % probe bound = cpm bound to dydroxyapatitecpm bound when no RNA          present/total cpm used in the assay                                      

The probes specific for the enteric Campylobacters, probes 2 and 4, werefurther tested and shown not to react with rRNAs of other organismsfound in the gastrointestinal tract.

                  TABLE 42                                                        ______________________________________                                        HYBRIDIZATION OF CAMPYLOBACTER PROBES 2 AND 4 TO                                ORGANISMS FOUND IN THE GASTROINTESTINAL TRACT.                                                     % Probe Bound (*)                                      Organism         ATCC#     Probe 2 Probe 4                                    ______________________________________                                        Citrobacter diversus                                                                           27156     0       0                                            Clostridium perfringens 13124 0 0                                             Enterobacter cloacae 13047 0 0                                                Klebsiella pneumoniae 23357 0 0.5                                             Proteus mirabilis 25933 0 0                                                   Serratia marcescens 13880 0 0                                                 Staphylococcus aureus e12600                                                  Staphylococcus epidermidis 14990 0 0.3                                        Streptococcus bovis 33317 0 0                                               ______________________________________                                         (*) % probe bound = cpm bound to hydroxyapatiscpm bound when no RNA           present/total cpm used in the assay                                      

EXAMPLE 13

Streptococci are gram positive, oxidase negative coccoid bacteria. Thegenus has been divided into 18 groups, A-R, on the basis ofgroup-specific carbohydrates. Group D streptococci are furthersubdivided into the enteroccocci (S. faecium, S. faecalis, S. avium andS. gallinarum and the non-enterococci S. bovis and S. eguinus. S.faecium, S. faecalis and S. avium are considered the medically importantenteroccocci. Some species of streptococcus are human pathogens; othersare normal flora in the mouth and intestine but are capable of causingdisease when introduced to other sites. Two examples are S. faecium andS. faecalis which are normally found in the intestine but may spread tocause bacteremia, wound infections, and as many as 10% of the urinarytract infections in the United States.

Current methods of detection of enterococci require culture of thespecimen for 18-72 hours followed by a battery of biochemical tests. Theoligonucleotide sequence shown below, when used in a hybridizationassay, accurately detects Streptococcus faecalis, S. avium, and S.faecium. The inventive probe does not cross react with otherStreptococci or Staphylococci which are very closely related in DNAhomology. (Kiepper-Baez, 1981, 1982, Schliefer 1984.) The currentinvention also reduces the number of tests which must be run on a sampleand greatly reduces the time to identification and thus, diagnosis. Thisrepresents a significant improvement over prior art methods.

The probe sequence was identified using a primer complementary to 16SrRNA with the sequence 5'-CCG CTT GTG CGG GCC CCC GTC AAT TC-3'. Thefollowing sequence was characterized and shown to be specific for threeenterocccci, S. faecium, S. faecalis and S. avium. The phylogeneticallynearest neighbors S. agalactiae, S. bovis, S. pneumoniae and S. pyogeneswere used for comparison with the sequences of interest.

1. TGC AGC ACT GAA GGG CGG AAA CCC TCC AAC ACT TA

The sequence is 35 bases in length and has a Tm of 72° C. It is capableof hybridizing in the region corresponding to bases 825-860 of E. coli16s rRNA. To demonstrate the reactivity and specificity of the probe, itwas used in a hybridization assay with purified RNA or RNA released fromcells. A suspension containing at least 10⁷ cells in 2% sodium dodecylsulfate was vortexed in the presence of glass beads. 0.1 ml ofsuspension was mixed with 0.1 ml of hybridization buffer (0.96M sodiumphosphate, pH 6.8, 0.002 M EDTA, 0.002 M EGTA) and incubated at 65° C.for 2 hours. After incubation, 5 ml of 2% hydoxyapatite, 0.12M sodiumphosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and the mixturewas incubated at 65° C. for 10 minutes. The sample was centrifuged andthe supernatant removed. Five ml of wash solution (0.12M phosphatebuffer, pH 6.8, 0.02% sodium dodecyl sulfate) was added and the sampleswere vortexed, centrifuged, and the supernatant removed. The amount ofradioactivity bound to the hydroxyapatite was determined byscintillation counting. Table 43 shows that the probe reacts well withS. faecium, S. faecalis, and S. avium, and does not react with otherclosely related organisms.

                  TABLE 43                                                        ______________________________________                                        HYBRIDIZATION OF THE ENTEROCOCCUS PROBE                                         TO CLOSELY RELATED ORGANISMS.                                                   Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Staphylococcus aureus                                                                           12600   1.4                                                   Streptococcus agalactiae 13813 1.5                                            Streptococcus avium 14025 22.7                                                Streptococcus bovis 33317 1.4                                                 Streptococcus faecalis 19433 45.3                                             Streptococcus faecium 19434 43.0                                              Streptococcus mitis  9811 1.5                                                 Streptococcus pneumoniae  6306 1.5                                            Streptococcus pyogenes 19615 1.3                                            ______________________________________                                    

EXAMPLE 14

Pseudomonads are gram-negative, nonsporeforming, nonfermentativebacilli. Pseudomonads are common inhabitants of soil and water andrarely infect healthy individuals. When the organisms encounter alreadycompromised patients, they can cause a variety of clinical syndromesincluding wound infections, post-surgical infections, septicemia, infantdiarrhea and respiratory and urinary tract infections. Members of thegenus Pseudomonas are particularly important to identify in a clinicalsample because of the resistance of the organisms to antibiotics.Nucleic acid homology studies have divided the genus into five homologyclasses known as RNA groups I-V. Eighty-three percent of all clinicalisolates of Pseudomonas are from RNA group I and Pseudomonas aeruginosais by far the most common species isolated.

Current methods of detection of pseudomonas require culture of a patientsample for 24-72 hours, followed by a battery of biochemical tests. Theoligonucleotide sequence below, when used in a hybridization assay,detects the clinically important group I pseudomonas. The presentinvention reduces the number of tests which must be run on a sample, andreduces the time to detection. This represents a significant improvementover prior art methods.

The sequence was obtained with a primer complementary to a conservedregion on 23S rRNA with the sequence 5'-CTT TCC CTC ACG GTA-3'. Thefollowing sequence was shown to detect group I pseudomonads:

1. CAG ACA AAG TTT CTC GTG CTC CGT CCT ACT CGA TT

The probe is 35 bases in length and has a Tm of 70° C. It is capable ofhybridizing to the RNA of group I Pseudomonas in the regioncorresponding to bases 365-405 of E. coli 23s rRNA. To demonstrate thereactivity and specificity of the probe, it was used in a hybridizationassay. ³² P-end-labeled oligonucleotide was mixed with RNA released fromat least 10⁷ organisms by standard methods in 0.48M sodium phosphate pH6.8, 1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM EGTA and incubated at65° C. for two hours. After incubation, the RNA:DNA hybrids were boundto hydroxyapatite as described for previous examples and theradioactivity bound was determined by scintillation counting. Table 44demonstrates that the probe reacted well with all 8 species of group Ipseudomonads that were tested. The probe did not react with RNA fromgroup II or group V organisms. A low reaction was seen with Pseudomonasacidovorans, a group III organism which represents <1% of all isolatesof nonfermentative bacilli from clinical samples. Table 45 demonstratesthat the probe does not react with other closely related organisms whichwere tested.

                  TABLE 44                                                        ______________________________________                                        HYBRIDIZATION OF PSEUDOMONAS GROUP I                                            PROBE TO PSEUDOMONAS RNAS                                                                                       % Probe*                                    Organism Group ATCC# Bound                                                  ______________________________________                                        Pseudomonas alcaligenes                                                                       I         14909   24                                            Pseudomonas aeruginosa I 10145 83                                             Pseudomonas denitrificans I 13867 83                                          Pseudomonas fluorescens I 13525 82                                            Pseudomonas mendocina I 25411 79                                              Pseudomonas pseudoalcaligenes I 17440 78                                      Pseudomonas putida I 12633 80                                                 Pseudomonas stutzeri I 17588 84                                               Pseudomonas cepacia II 25416 0                                                Pseudomonas pickettii II 27511 1.0                                            Pseudomonas acidovorans III 15668 11                                          Pseudomonas maltophilia V 13637 0.2                                         ______________________________________                                         *% Probe Bound = counts bound when RNA present  counts bound when no RNA      present/total counts used in the assay                                   

                  TABLE 45                                                        ______________________________________                                        HYBRIDIZATION OF PSEUDOMONAS GROUP I                                            PROBE TO RNAs OF CLOSELY RELATED ORGANISMS                                                         % Probe*                                               Organism           ATCC#   Bound                                              ______________________________________                                        Acinetobacter calcoaceticus                                                                      23055   1.6                                                  Legionella pneumophila 33155 0.6                                              Moraxella phenylpyruvica 23333 0.3                                            Morganella morganii 25830 0                                                   Vibrio parahaemolyticus 17802 0.6                                           ______________________________________                                         *% Probe Bound = counts bound when RNA present  count bound when no RNA       present/total counts used in the assay                                   

EXAMPLE 15

Examples 15-18 disclose probes for the Enterobacteriaceae, all of whichare highly related at the DNA level. Even fewer differences exist at therRNA level. For example, Proteus vulgaris 16s rRNA is 93% homologous toE. coli. These factors illustrate the difficulties associated withmaking rRNA probes specific for this group of organisms. Nevertheless,we have invented probes for Enterobacter cloacae, Proteus mirabilis,Salmonella and E. coli.

Members of the genus Enterobacter are motile, gram negative,non-sporeforming bacilli which belong in the family Enterobacteriaceae.The genus is a large and heterogeneous group. Eight species have beendefined but only 5 are clinically significant. Enterobacter cloacae andE. aerogenes are the most common isolates and are associated withgenitourinary, pulmonary, blood, central nervous system and soft tissueinfections in humans.

The current method for identifying Enterobacter cloacae from patientsamples involves culture of the specimen on agar plates for 18-24 hours,followed by a battery of biochemical tests. The oligonucleotide sequencedescribed below, when used as a probe in a nucleic acid hybridizationassay, accurately identifies Enterobacter cloacae. The present inventionreduces the number of tests which must be run on a sample, the time toidentification and therefore, diagnosis, and thus represents asignificant improvement over prior art methods.

The probe specific for Enterobacter cloacae was obtained with a primercomplementary to a conserved region of 23S rRNA with the sequence 5'-CAGTCA GGA GTA TTT AGC CTT-'3.

The following sequence was characterized and shown to be specific for E.cloacae. The phylogenetically nearest neighbors Escherichia coli,Klebsiella pneumoniae, Proteus vulgaris, Salmonella enteritidis, andCitrobacter freundii were used as comparisons with the sequence of E.cloacae.

1. GTG TGT TTT CGT GTA CGG GAC TTT CAC CC

The probe is 29 bases in length and has a Tm of 68° C. It is capable ofhybridizing to RNA of E. cloacae in the region corresponding to bases305-340 of E. coli 23s rRNA. To demonstrate the reactivity andspecificity of the probe for E. cloacae, it was used in a hybridizationassay. ³² P-end-labeled oligonucleotide probe was mixed with RNAreleased from at least 10⁷ organisms in 1% sodium dodecyl sulfate, 0.48Msodium phosphate, pH 6.8 (0.2 ml final volume) and incubated at 60° C.for 2 hours. Following incubation, 5 ml of 2% hydroxyapatite, 0.12Msodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and themixture incubated at 60° C. for 10 minutes. The sample was centrifugedand the supernatant removed. Five ml of wash solution (0.12M sodiumphosphate, pH 6.8, 0.02% sodium dodecyl sulfate) was added, the samplevortexed, centrifuged and the supernatant removed. The amount ofradioactivity bound to the hydroxyapatite was determined byscintillation counting. The results are shown in Table 46 anddemonstrates that the probe reacts well with E. cloacae and does notreact with the RNA of closely related organisms.

                  TABLE 46                                                        ______________________________________                                        HYBRIDIZATION OF ENTEROBACTER CLOACAE PROBE                                     TO CLOSELY RELATED ORGANISMS                                                                              % Probe                                           Organisms Name ATCC# Bound                                                  ______________________________________                                        Citrobacter freundii                                                                             8090   1.8                                                   Enterobacter aerogenes 13048 1.4                                              Enterobacter cloacae 13047 27.                                                Escherichia coli 11775 1.0                                                    Klebsiella pneumoniae 13883 1.7                                               Proteus mirabilis 29906 0.9                                                   Proteus vulgaris 13315 0.6                                                    Providencia stuartii 29914 1.1                                              ______________________________________                                    

Table 47 shows that the probe does not react with the RNA of organismsfound in urine.

                  TABLE 47                                                        ______________________________________                                        HYBRIDIZATION OF ENTEROBACTER CLOACAE                                           PROBE TO ORGANISMS FOUND IN URINE.                                                                         % Probe                                          Organisms Name ATCC# Bound                                                  ______________________________________                                        Candida albicans   18804   0.8                                                  Candida krusei 34135 0.8                                                      Candida parapsilosis 22019 0.9                                                Candida tropicalis  750 1.1                                                   Pseudomonas aeruginosa 10145 1.0                                              Serratia marcescens 13880 1.6                                                 Staphylococcus aureus 12600 1.7                                               Staphylococcus epidermidis 14990 1.4                                          Streptococcus agalactiae 13813 2.5                                            Streptococcus faecium 19434 1.5                                               Torulopsis glabrata  2001 0.9                                               ______________________________________                                    

EXAMPLE 16

Members of the genus Proteus are motile, gram negative, non-sporeformingbacilli which belong in the family Enterobacteriaceae. Four species ofProteus have been described and three of them, Proteus mirabilis, P.vulgaris, and P. penneri, cause human disease.

The most common type of proteus infection involves the urinary tract,but septicemia, pneumonia and wound infections also occur. Proteusmirabilis is the species most often isolated and may account for up to10% of all acute, uncomplicated urinary tract infections. Species,rather than genus level identification of the causative organism isdesirable because of differential antibiotic susceptibility among thespecies.

The current method for identifying Proteus mirabilis from patientsamples involves culture of the specimen on agar plates for 18-24 hours,followed by a battery of biochemical tests. The oligonucleotide sequencedescribed below, when used as a probe in a nucleic acid hybridizationassay, accurately identifies Proteus mirabilis. The present inventionreduces the number of tests which must be run on a sample, the time toidentification and therefore, diagnosis and treatment. This represents asignificant improvement over prior art methods.

The probe specific for Proteus mirabilis was obtained with a primercomplementary to a conserved region of 23S rRNA with the sequence 5'-CAGTCA GGA GTA TTT AGC CTT-3'.

The following sequence was characterized and shown to be specific for P.mirabilis. The phylogenetically nearest neighbors Escherichia coli,Klebsiella pneumoniae, Proteus vulgaris and Salmonella enteritidis wereused as comparisons with the sequence of Proteus mirabilis.

1. CCG TTC TCC TGA CAC TGC TAT TGA TTA AGA CTC

This probe is capable of hybridizing to the RNA of P. mirabilis in theregion corresponding to base 270-305 of E. coli 23s rRNA. The probe is33 bases in length and has a Tm of 66° C. To demonstrate the reactivityand specificity of the probe for P. mirabilis, it was used in ahybridization assay. ³² P-end-labeled oligonucleotide probe was mixedwith RNA released from at least 10⁷ organisms in 1% sodium dodecylsulfate, 0.48M sodium phosphate, pH 6.8, 1 mM EDTA, 1 mM EGTA (0.2 mlfinal volume) and incubated at 64° C. for 2 hours. Following incubation,5 ml of 2% hydroxyapatite, 0.12M sodium phosphate pH 6.8, 0.02% sodiumdodecyl sulfate was added and the mixture incubated at 64° C. for 10minutes. The sample was centrifuged and the supernatant removed. Five mlof wash solution (0.12 M sodium phosphate, pH 6.8, 0.02% sodium dodecylsulfate) was added, the sample vortexed, centrifuged and the supernatantwas removed. The amount of radioactivity bound to the hydroxyapatite wasdetermined by scintillation counting. The results are shown in Table 48and demonstrate that the probe reacts well with P. mirabilis and doesnot react with 27 other closely related bacteria. Table 49 shows thatthe probe does not react with 24 other phylogenetically diverse bacteriaand two yeasts tested in the same manner as the organisms in Table 48.

                  TABLE 48                                                        ______________________________________                                        HYBRIDIZATION OF PROTEUS MIRABILIS PROBE                                        TO CLOSELY RELATED ORGANISMS                                                                               % Probe                                          Organism Name ATCC# Bound                                                   ______________________________________                                        Citrobacter diversus                                                                             27156   1.1                                                  Citrobacter freundii  8090 1.1                                                Citrobacter freundii  6750 1.0                                                Enterobacter aerogenes 13048 1.0                                              Enterobacter agglomerans 27155 1.0                                            Enterobacter cloacae e13047 1.1                                               Enterobacter gergoviae 33028 1.0                                              Enterobacter sakazakii 29544 1.1                                              Escherichia coli 10798 1.2                                                    Escherichia coli 11775 1.2                                                    Escherichia coli 29417 1.2                                                    Klebsiella oxytoca 13182 1.0                                                  Klebsiella ozaenae 11296 1.1                                                  Klebsiella planticola 33531 0.9                                               Klebsiella pneumoniae 13883 1.3                                               Klebsiella pneumoniae 23357 1.1                                               Klebsiella rhinoscleromatis 13884 1.2                                         Klebsiella terrigena 33257 1.1                                                Klebsiella trevisanii 33558 1.0                                               Kluyvera ascorbata 33433 0.9                                                  Proteus mirabilis 25933 69.0                                                  Proteus penneri 33519 2.5                                                     Proteus vulgaris 13315 1.7                                                    Providencia alcalifaciens  9886 1.1                                           Providencia rettgeri 29944 1.3                                                Providencia stuartii 29914 1.1                                                Salmonella arizonae 29933 1.1                                                 Salmonella enteritidis 13076 0.8                                            ______________________________________                                    

                  TABLE 49                                                        ______________________________________                                        HYBRIDIZATION OF PROTEUS MIRABILIS PROBE TO                                     PHYLOGENETICALLY DIVERSE ORGANISMS                                                                          % Probe                                         Organism Name ATCC# Bound                                                   ______________________________________                                        Acinetobacter calcoaceticus                                                                       33604   0.8                                                 Bacillus subtilis  6051 1.2                                                   Bacteroides fragilis 23745 0.9                                                Branhamella catarrhalis 25238 0.7                                             Campylobacter jejuni 33560 1.0                                                Candida krusei 34135 0.8                                                      Chromobacterium violaceum 29094 1.1                                           Clostridium perfringens 13124 0.9                                             Deinoccoccus radiodurans 35073 0.8                                            Derxia gummosa 15994 0.8                                                      Hafnia alvei 13337 0.9                                                        Morganella morganii 25830 0.9                                                 Pseudomonas aeruginosa 10145 1.0                                              Pseudomonas cepacia 17762 0.9                                                 Rahnella aquatilis 33071 0.9                                                  Rhodospirillum rubrum 11170 0.8                                               Serratia marcescens 13880 0.9                                                 Serratia odorifera 33077 0.9                                                  Staphylococcus aureus e12600 0.8                                              Staphylococcus epidermidis 14990 0.8                                          Streptococcus mitis  9811 0.8                                                 Streptococcus pneumoniae e6306 0.9                                            Torulopsis glabrata  2001 0.9                                                 Vibrio parahaemolyticus 17802 0.8                                             Xanthomonas maltophilia 13637 1.1                                             Yersinia enterocolitica  9610 0.8                                           ______________________________________                                    

Example 17

Members of the genus Salmonella are motile, gram negative,non-sporeforming bacilli which belong in the family Enterobacteriaceae.All salmonellae are highly related and some microbiologists considerthem to be one species. Five subgroups have been identified usingnucleic acid homology studies and over 1400 different serotypes havebeen described. All serotypes have been implicated in human entericdisease ranging from self-limited gastroenteritis with mild symptoms, tosevere gastroenteritis with bacteremia, to typhoid fever, a potentiallylife-threatening illness. S. cholerasuis, S. paratyphi A and S. typhiare the serotypes most often associated with severe disease andbacteremia. Diagnosis of Salmonella-induced enteritis is dependent upondetection of the organism in stool samples. Because infection occursprimarily by ingestion of contaminated milk, food and water, methods foridentifying Salmonella in these products before release to consumers iscritical.

Current methods for detection of members of the genus Salmonella involveculture of the specimen for 1-3 days on selective media followed by abattery of biochemical tests. Often an enrichment step is needed toisolate Salmonella from clinical samples or food products. Theoligonucleotide sequences shown below, when used in a hydridizationassay, accurately identify members of the genus Salmonella. The presentinventive probes are specific for all members of the genus and do notreact with the other closely related Enterobacteriaceae genera. Theseinventive probes reduce the number of tests which must be run on asample and greatly reduce the time to identification. This represents asignificant improvement over prior art methods.

The probes specific for the genus Salmonella were obtained with twoprimers complementary to 16S and 23S rRNA. Sequence 1 was obtained usinga 16S primer with the sequence 5' TTA CTA GCG ATT CCG ACT TCA 3'.Sequence 2 was obtained using a 23S primer with the sequence 5' CAG TCAGGA GTA TTT AGC CTT 3'. The following sequences were characterized andshown to be specific for the genus Salmonella:

    1.   CTC CTT TGA GTT CCC GAC CTA ATC GCT GGC                                     - 2.  CTC ATC GAG CTC ACA GCA CAT GCG CTT TTG TGT A                    

Sequence 1, from 16S rRNA, is 30 bases in length and has a Tm of 73° C.Sequence 2, from 23S rRNA, is 34 bases long and has a Tm of 71° C. Theseprobes are capable of hybridizing in the regions corresponding to bases1125-1155 of E. coli 16s rRNA and 335-375 of E. coli 23s rRNA,respectively. To demonstrate the reactivity and specificity of probe 1for members of the genus Salmonella, ³² P-end-labeled oligonucleotidewas tested as a probe in a hybridization reaction. Purified RNA, or RNAreleased from at least 10⁷ organisms by standard methods, was mixed with1 ml hybridization buffer (final concentration 43% diisobutylsulfosuccinate, 60 mM sodium phosphate pH 6.8, 1 mM EDTA, 1 mM EGTA) andincubated at 72° C. for 2-12 hours. Following incubation, 5 ml ofseparation solution (2% hydroxyapatite, 0.12 M sodium phosphate, pH 6.8,0.02% sodium dodecyl sulfate) was added and the sample were mixed,incubated at 72° C. for 5 minutes, centrifuged and the supernatantsremoved. Four ml of wash solution (0.12 M sodium phosphate pH 6.8, 0.02%sodium dodecyl sulfate) was added and the samples were vortexed,centrifuged, and the supernatants removed. The amount of radioactivitybound to the hydroxyapatite was determined by scintillation counting.The results shown in Table 50 indicate that a combination of the twoprobes hybridized to the 5 subgroups of Salmonella and to all 31 of theserotypes which were tested.

                  TABLE 50                                                        ______________________________________                                        HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2                                      TO MEMBERS OF THE GENUS SALMONELLA                                                                             % Probe Bound                              Subgroup                                                                              Organism         ATCC#   Probe 1                                                                             Probe 2                                ______________________________________                                        I       Salmonella choleraesuis                                                                        10708   24    40                                       I Salmonella enteritidis 13076 15 67                                          I Salmonella paratyphi A 9150 1.4 70                                          I Salmonella sp. serotype 9270 40 26                                           anatum                                                                       I Salmonella sp. serotype 12007 54 35                                          cubana                                                                       I Salmonella sp. serotype give 9268 12 40                                     I Salmonella sp. serotype 8326 53 33                                           heidelberg                                                                   I Salmonella sp. serotype 11646 36 46                                          illinois                                                                     I Salmonella sp. serotype 8387 35 32                                           montevideo                                                                   I Salmonella sp. serotype 29628 52 34                                          newington                                                                    I Salmonella sp. serotype 6962 3.4 36                                          newport                                                                      I Salmonella sp. serotype 15787 34 39                                          putten                                                                       I Salmonella sp. serotype 9712 28 30                                           saintpaul                                                                    I Salmonella sp. serotype 8400 38 43                                           senftenberg                                                                  I Salmonella sp. serotype 12004 29 29                                          simsbury                                                                     I Salmonella sp. serotype 15791 34 30                                          sloterdijk                                                                   I Salmonella sp. serotype 8391 32 41                                           thompson                                                                     I Salmonella sp. serotype 15611 35 2.6                                         vellore                                                                      I Salmonella typhi 19430 7.0 21                                               I Salmonella typhimurium 14028 69 69                                          II Salmonella salamae 6959 3.0 46                                             II Salmonella sp. serotype 15793 6.6 30                                        maarssen                                                                     III Salmonella arizonae 33952 2.9 38                                          III Salmonella arizonae 12324 5.5 42                                          III Salmonella arizonae 29933 2.3 62                                          III Salmonella arizonae 29934 63 12                                           III Salmonella arizonae 12323 4.0 39                                          III Salmonella arizonae 12325 51 1.9                                          IV Salmonella sp. serotype 15783 5.8 8.0                                       harmelen                                                                     IV Salmonella sp. serotype 29932 7.5 40                                        ochsenzoll                                                                   V Salmonella sp. serotype cdc1319 60 1.8                                       bongor                                                                     ______________________________________                                    

The specificity of the probes for members of the genus Salmnonella wasdemonstrated with hybridization reactions containing RNA from organismsclosely related to Salmonella. The results are shown in Table 51.

                  TABLE 51                                                        ______________________________________                                        HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2                                      TO RNA OF CLOSELY RELATED ORGANISMS                                                                % Probe Bound                                          Organism         ATCC#     Probe 1 Probe 2                                    ______________________________________                                        Citrobacter freundii                                                                            6750     2.2     0                                            Edwardsiella tarda 15947 0 0                                                  Enterobacter agglomerans 27155 0.6 0                                          Enterobacter cloacae 13047 0 0                                                Enterobacter sakazakii 29544 0 0                                              Escherichia coli 10798 0 0                                                    Escherichia coli 29417 0 0                                                    Klebsiella pneumoniae 23357 0.7 0                                             Kluyvera ascorbata 33433 0 0.5                                                Proteus mirabilis 25933 0.2 0                                                 Shigella flexneri 29903 0 0                                                 ______________________________________                                         * % Probe Bound = counts bound to dydroxyapatite  counts bound when no RN     present/total counts used in assay                                       

Table 52 shows that Salmonella probes 1 and 2 do not hybridize tophylogenetically diverse organisms.

                  TABLE 52                                                        ______________________________________                                        HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2 TO                                   RNA OF A PHYLOGENETIC CROSS SECTION OF ORGANISMS                                                   % Probe Bound*                                         Organism          ATCC#    Probe 1 Probe 2                                    ______________________________________                                        Acinetobacter calcoaceticus                                                                     33604    1.1     0.1                                          Bacillus subtilis  6051 0 0.5                                                 Bacteroides fragilis 23745 0.1 0                                              Branhamella catarrhalis 25238 0.9 0                                           Campylobacter jejuni 33560 0 0.2                                              Candida krusei 34135 0.4 0.3                                                  Chromobacterium violaceum 29094 1.7 0                                         Clostridium perfringens 13124 0.3 0                                           Deinococcus radiodurans 35073 1.6 0.1                                         Derxia gummosa 15994 1.2 0                                                    Hafnia alvei 13337 1.8 0                                                      Morganelli morganii 25830 0 1.1                                               Pseudomonas aeruginosa 10145 0.5 0.7                                          Pseudomonas cepacia 17762 0 0                                                 Pseudomonas maltophilia 13637 1.9 0                                           Rahnella aquatilis 33071 1.2 0.3                                              Rhodospirillum rubrum 11170 0.9 0                                             Serratia marcescens 13880 0 0                                                 Serratia odorifera 33077 2.6 0.2                                              Staphylococcus aureus e12600 0.2 0                                            Staphylococcus epidermidis 14990 0 0                                          Streptococcus nitis  9811 1.2 0.7                                             Streptococcus pneumoniae e6306 0 0                                            Torulopsis glabrata  2001 0 0                                                 Vibrio parahaemolyticus 17802 0 0.2                                           Yersinia enterocolitica  9610 0 0                                           ______________________________________                                         *% Probe Bound = Counts bound to hydroxyapatite  counts bound when no RNA     present/total counts used in assay                                       

Example 18

Escherichia coli is a gram negative, nonsporeforming bacillus whichbelongs in the family Enterobacteriaceae. Five species of Escherichiahave been described: E. coli, which accounts for >99% of the clinicalisolates, E. hermanii, E. blattae, E. vulneris and E. fergusonii. E.coli is a leading cause of urinary tract infections, bactermia andneonatal meningitidis, and can cause a type of gastroenteritis known astraveller's diarrhea.

The current method for identifying E. coli from patient samples involvesculture of the specimen on agar plates for 18-72 hours, followed by abattery of biochemical tests on isolated colonies. The oligonucleotidesequence described below, when used as a probe in a nucleic acidhybridization assay, accurately detects E. coli even in the presence ofother organisms. The present invention reduces the number of tests whichmust be run on a sample and reduces the time to identification andtherefore diagnosis and treatment. This represents a significantimprovement over prior art methods.

The probe specific for E. coli was derived from the published E colisequence (Brosius, et al. Proc. Natl. Acad. Sci, U.S.A. 75:4801-4805(1978)), using Proteus vulgaris (Carbon, et al., Nuc, Acids Res.9:2325-2333 (1981)), Klebsiella pneumoniae, Salmonella enteritidis,Enterobacter gergoviae and Citrobacter freundii for comparison. Theprobe sequence is shown below.

1. GCA CAT TCT CAT CTC TGA AAA CTT CCG TGG

It hybridizes to RNA of E. coli in the region of 995-1030 of 16s rRNA.The probe is 30 bases in length and has a T_(m) of 66° C. To demonstratethe reactivity and specificity of the probe for E. coli, it was used ina hybridization assay. ³² p-end-labeled oligonucleotide probe was mixedwith two unlabeled oligonucleotides of sequence 5'-TGG ATG TCA AGA CCAGGT AAG GTT CTT CGC GTT GCA TCG-3' and 5'-CTG ACG ACA GCC ATG CAG CACCTG TCT CAC GGT TCC CGA AGG CA-3' and with purified RNA, or RNA releasedfrom cells with detergent and heat, in 1% sodium dodecyl sulfate (SDS),0.48 M sodium phosphate pH 6.8, 1 mM EDTA, 1 mM EGTA (0.2 ml finalvolume) and incubated at 60° C. for 2 hours. Following incubation, 5 mlof 2% hydroxyapatite, 0.12 M sodium phosphate pH 6.8, 0.02% sodiumdodecyl sulfate was added and the mixture incubated at 60° C. for 10minutes. The sample was centrifuged and the supernatant removed. Five mlof wash solution (0.12 M sodium phosphate, pH 6.8, 0.02% sodium dodecylsulfate) was added, the sample vortexed, centrifuged and the supernatantwas removed. The amount of radioactivity bound to the hydroxyapatite wasdetermined by scintillation counting.

An example of a use for this probe would be to detect E. coli in urinesamples. Table 53 shows that the probe detects 7 out of 8 strains of E.coli tested. The probe also reacts with E. fergusonii, an organism whichwould only rarely be found in urine.

Table 54 shows that the probe does not react with any other genus testedexcept Shigella, another organism rarely isolated from urine. Theseresults show that the probe will be useful in detecting E. coli fromurine samples.

                  TABLE 53                                                        ______________________________________                                        HYBRIDIZATION OF E. coli TO ESCHERICHIA SPECIES                                    Organism       ATCC#   % Probe Bound                                     ______________________________________                                        Escherichia coli                                                                              10798   70                                                      E. coli 11775 67                                                              E. coli 23722 58                                                              E. coli 25404 68                                                              E. coli 25922 55                                                              E. coli 29417 72                                                              E. coli 33780 0.8                                                             E. coli 35150 45                                                              E. fergusonii 35469 55                                                        E. hermanii 33650 0.7                                                         E. vulneris 33821 0.8                                                       ______________________________________                                    

                  TABLE 54                                                        ______________________________________                                        HYBRIDIZATION OF THE E. coli PROBE TO                                           CLOSELY RELATED ORGANISMS                                                       Organism          ATCC#   % Probe Bound                                   ______________________________________                                        Citrobacter freundii                                                                             6750   0.8                                                   Citrobacter freundii  8090 0.9                                                Citrobacter freundii 29221 0.6                                                Citrobacter freundii 33128 0.6.                                               Enterobacter aerogenes 13048 1.2                                              Enterobacter agglomerans 27155 0.9                                            Enterobacter cloacae 13047 0.9                                                Enterobacter gergoviae 33023 0.7                                              Enterobacter sakazakii 29544 0.6                                              Klebsiella oxytoca 13182 0.7                                                  Klebsiella pneumoniae 13883 0.7                                               Proteus mirabilis 29906 0.7                                                   Proteus vulgaris 13315 0.8                                                    Shibella boydii  8700 76                                                      Shigella dysenteriae 13313 0.8                                                Shigella flexneri 29903 71                                                    Shigella sonnei 29930 75                                                    ______________________________________                                    

Example 19

The bacteria encompass a morphologically and physiologically diversegroup of unicellular organisms which occupy most natural environments.Although many bacteria are harmless or beneficial to their environmentor host, some are harmful and cause disease. The presence of anybacteria in some locations is undesirable or indicative of disease(e.g., culture media, pharmaceutical products, body fluids such asblood, urine or cerebrospinal fluid, and tissue biopsies). Low levels ofbacteria are considered acceptable in other products such as drinkingwater and food products. Accordingly, there is a need for a means fordetecting and quantitating bacteria in a sample.

The current method of detection and quantitation of total bacteria in asample requires culture on multiple types of media under differentconditions of temperature and atmosphere. To date, no single test existsto detect or quantitate all bacteria. The oligonucleotide sequencesshown below, when used in a hybridization assay, detect a broadphylogenetic cross section of bacteria. The present invention reducesthe number of tests which need to be performed and also reduces the timerequired for the assay. Comparison of the hybridization results from anunknown sample to a set of standards will allow some quantitation of thenumber of bacteria present. This represents a significant improvementover prior art methods.

The bacterial probes were designed following examination of publishedsequences of rRNA and sequences determined at Gen-Probe. The sequencesused for the comparison include Agrobacterium tumefaciens (Yang et al.,Proc. Natl. Acad. Sci. U.S.A., 82:4443, (1985), Anacystis nidulans(Tomioka and Sugiura. Mol. Gen, Genet. 191:46, (1983), Douglas andDoolittle Nuc. Acids Res. 12:3373, (1984), Bacillus subtilis (Green etal., Gene 37:261. (1985), Bacillus stearothermophilus (Kop et al., DNA3:347, (1984), Bacteroides fragilis (Weisburg et al., J. Bacteriol.164:230, (1985), Chlamydia psittaci (Weisburg et al., J. Bacteriol.167:570. (1986)), Desulfoyibrio desulfuricans (Oyaizu and Woese, System.Appl. Microbiol. 6:257, (1985); Escherichia coli, (Brosius et al., Proc.Natl. Acad. Sci. U.S.A. 77:201, (1980); Flavobacterium heparinum(Weisburg et al., J. Bacteriol. 164:230, (1985); Heliobacterium chlorum(Woese et al., Science 229:762, (1985); Mycoplasma PG50 (Frydenberg andChristiansen, DNA 4:127, (1985); Proteus vulgaris (Carbon et al., Nuc.Acids Res. 9:2325, (1981); Pseudomonas testosteroni (Yang et al., Proc.Natl. Acad. Sci. U.S.A. 82:4443, (1985); Rcchalimaea guintana (Weisburget al., Science 230:556, (1985); Saccharomyces cerevisiae (Rubstov etal., Nuc. Acids Res. 8:5779, (1980); Georgiev et al., Nuc. Acids Res.9:6953, (1981); and human (Torczynski et al., DNA 4:283, (1985);Gonzalez et al., Proc. Natl. Acad. Sci. U.S.A. 82:7666, (1985)).

The following sequences were shown to hybridize to a broad phylogeneticcross section of bacteria and not to yeast or human rRNA:

    1.   CCA CTG CTG CCT CCC GTA GGA GTC TGG GCC                                     - 2. CCA GAT CTC TAC GCA TTT CAC CGC TAC ACG TGG                              - 3. GCT CGT TGC GGG ACT TAA CCC AAC AT                                       - 4. GGG GTT CTT TTC GCC TTT CCC TCA CGG                                      - 5. GGC TGC TTC TAA GCC AAC ATC CTG                                          - 6. GGA CCG TTA TAG TTA CGG CCG CC                                           - 7. GGT CGG AAC TTA CCC GAC AAG GAA TTT CGC TAC C                     

Probe 1 is 30 bases long and has a Tm of 70° C. Probe 2 is 33 bases longand has a Tm of 69° C. Probe 3 is 26 bases long and has a Tm of 67° C.Probe 4 is 27 bases long and has a Tm of 69° C. Probe 5 is 24 bases longand has a Tm of 66° C. Probe 6 is 23 bases long and has a Tm of 62° C.Probe 7 is 34 bases long and has a Tm of 66° C. Probes 1-3 hybridize to16S rRNA in the following regions, respectively, (corresponding to E.coli bases) 330-365; 675-715; and 1080-1110. Probes 4-7 hybridize to 23SrRNA in the following regions, respectively, (corresponding to E. colibases) 460-490; 1050-1080; and 1900-1960 (probes 6 and 7). Theoligonucleotides interact with regions on the rRNA which are highlyconserved among eubacteria. This means that they can be used asbacterial probes in a hybridization assay. A second use is as a tool toobtain rRNA sequence. For example, an oligonucleotide can be hybridizedto the rRNA of interest and extended with reverse transcriptase. Thesequence of the resulting DNA can be determined and used to deduce thecomplementary rRNA sequence as described in the Detailed Description ofthe Invention.

One application of the invention is to detect bacteria in urine(bacteriuria). To demonstrate the reactivity and specificity of theprobes for bacteria found in urine, they were used in hybridizationassays. ³² P-end-labeled or ¹²⁵ I-labeled oligonucleotide probes weremixed with RNA released from cells by standard methods (e.g, the sonicdisruption techniques described in Murphy et al., U.S. patentapplication Ser. No. 841,860, detergent with glass beads, or enzymaticlysis). Probe was mixed with RNA in 0.48 M sodium phosphate, pH 6.8, 1mM EDTA, 1 mM EGTA, 1% sodium dodecyl sulfate (0.2 ml final volume) andhybridized at 60° C. for 2 hours. Five ml of 2% hydroxyapatite, 0.12 Msodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and themixture incubated at 60° C. for 10 minutes. The mixture was centrifugedand the supernatant removed. Five ml of wash solution (0.12 M sodiumphosphate, pH 6.8, 0.02% sodium dodecyl sulfate) was added and thesample was mixed, centrifuged and the supernatant removed. The amount ofradioactivity bound to the hydroxyapatite was determined byscintillation counting. Tables 55-68 demonstrate the specificity ofthese probes and show that a combination of probes could be used todetect all bacteria wich have been tested.

Table 55 shows that probe 1 hybridizes to the RNA of bacteria commonlyisolated frome urine and does not detect yeast RNA. Table 56 shows thatprobe 1 detects phylogenetically diverse bacteria and does not hybridizeto human RNA.

                  TABLE 55                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 1                                              TO RNA OF ORGANISMS FOUND IN URINE                                                                         % Probe*                                         Organism ATCC# Bound                                                        ______________________________________                                        Candida albicans   18804   2.6                                                  Candida krusei 34135 2.2                                                      Candida parapsilosis 22019 2.9                                                Candida tropicalis  750 2.5                                                   Citrobacter freundii  8090 69                                                 Enterobacter aerogenes 13048 70                                               Enterobacter cloacae 13047 71                                                 Escherichia coli 11775 67                                                     Klebsiella oxytoca 13182 70                                                   Klebsiella pneumoniae 13883 72                                                Morganelia morganii 25830 66                                                  Proteus mirabilis 29906 71                                                    Proteus vulgaris 13315 67                                                     Providencia stuartii 29914 69                                                 Pseudomonas aeruginosa 10145 76                                               Pseudomonas fluorescens 13525 73                                              Serratia marcescens 13880 66                                                  Staphylococcus aureus 12600 57                                                Staphylococcus epidermidis 14990 68                                           Streptococcus agalactiae 13813 66                                             Streptococcus faecalis 19433 51                                               Streptococcus faecium 19434 53                                                Torulopsis glabrata  2001 2.3                                                 Ureaplasma urealyticum 27618 54                                             ______________________________________                                    

                  TABLE 56                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 1 TO RNAs OF A CROSS                           SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS.                                                              % Probe*                                        Organism ATCC# Bound                                                        ______________________________________                                        Acinetobacter calcoaceticus                                                                       23055   65                                                  Bacillus subtilis  6051 73                                                    Bacteroides fragilis 23745 61                                                 Branhamella catarrhalis 25238 72                                              Campylobacter jejuni 33560 64                                                 Chlamydia trachomatis VR878 14                                                Chromabacterium violaceum 29094 71                                            Clostridium perfringens 13124 74                                              Corynebacterium xerosis  373 38                                               Deinococcus radiodurans 35073 47                                              Derxia gummosa 15994 65                                                       Gardnerella vaginalis 14018 67                                                Hafnia alvei 13337 60                                                         Lactobacillus acidophilus  4356 56                                            Moraxella osloensis 19976 61                                                  Mycobacterium smegmatis 14468 47                                              Mycoplasma hominis 14027 58                                                   Neisseria gonorrhoeae 19424 58                                                Rahnella aquatilis 33071 74                                                   Rhodospirillum rubrum 11170 73                                                Vibrio parahaemolyticus 17802 75                                              Human  2.5                                                                  ______________________________________                                    

Table 57 shows that Probe 2 hybridizes to the RNA of bacteria commonlyfound in urine except Ureaplasma urealyicum and does not hybridize toyeast rRNA.

                  TABLE 57                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 2                                              TO RNA OF ORGANISMS FOUND IN URINE                                                                         % Probe*                                         Organism ATCC# Bound                                                        ______________________________________                                        Candida albicans   18804   2.5                                                  Candida krusei 34135 1.8                                                      Candida parapsilosis 22019 1.6                                                Candida tropicalis  750 1.4                                                   Citrobacter freundii  8090 61                                                 Enterobacter aerogenes 13048 57                                               Enterobacter cloacae 13047 61                                                 Escherichia coli 11775 67                                                     Klebsiella oxytoca 13192 67                                                   Klebsiella pneumoniae 13883 51                                                Morganella morganii 25830 69                                                  Proteus mirabilis 29906 69                                                    Proteus vulgaris 13315 69                                                     Providencia stuartii 29914 66                                                 Pseudomonas aeruginosa 10145 59                                               Pseudomonas fluorescens 13525 58                                              Serratia marcescens 13880 64                                                  Staphylococcus aureus 12600 60                                                Staphylococcus epidermidis 14990 60                                           Streptococcus agalactiae 13813 54                                             Streptococcus faecalis 19433 37                                               Streptococcus faecium 19434 58                                                Torulopsis glabrata  2001 1.5                                                 Ureaplasma urealyticum 27618 3.2                                            ______________________________________                                    

Table 58 shows that probe 2 detects phylogenetically diverse bacteriaand does not hybridize to human rRNA.

                  TABLE 58                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 2 TO RNAs OF A CROSS                           SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS.                                                              % Probe*                                        Organism ATCC# Bound                                                        ______________________________________                                        Acinetobacter calcoaceticus                                                                       23055   76                                                  Bacillus subtilis  6051 75                                                    Bacteroides fragilis 23745 2.0                                                Branhamella catarrhalis 25238 70                                              Campylobacter jejuni 33560 2.5                                                Chlamydia trachomatis VR878 16                                                Chromobacterium violaceum 29094 61                                            Clostridium perfringens 13124 66                                              Corynebacterium xerosis  373 3.8                                              Deinococcus radiodurans 35073 6.0                                             Derxia gummosa 15994 61                                                       Gardnerella vaginalis 14018 2.0                                               Hafnia alvei 13337 72                                                         Lactobacillus acidophilus  4356 50                                            Moraxella osloensis 19976 64                                                  Mycobacterium smegmatis 14468 19                                              Mycoplasma hominis 14027 34                                                   Neisseria gonorrhoeae 19424 71                                                Rahnella aauatilis 33071 77                                                   Rhodospirillum rubrum 11170 1.5                                               Vibrio parahaemolyticus 17802 73                                              Yersinia enterocolitica  9610 76                                              Human  2.0                                                                  ______________________________________                                    

Table 59 shows that probe 3 hybridizes to the RNA of bacteria commonlyfound in urine and does not detect yeast rRNA.

                  TABLE 59                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 3                                              TO RNA OF ORGANISMS FOUND IN URINE                                                                         % Probe*                                         Organism ATCC# Bound                                                        ______________________________________                                        Candida albicans   18804   1.4                                                  Candida krusei 34135 1.5                                                      Candida parapsilosis 22019 2.2                                                Candida tropicalis  750 2.6                                                   Citrobacter freundii  8090 79                                                 Enterobacter aerogenes 13048 40                                               Enterobacter cloacae 13047 44                                                 Escherichia coli 11775 67                                                     Klebsiella oxytoca 13182 38                                                   Klebsiella pneumoniae 13883 45                                                Morganella morganii 25830 57                                                  Proteus mirabilis 29906 40                                                    Proteus vulgaris 13315 51                                                     Providencia stuartii 29914 54                                                 Pseudomonas aeruginosa 10145 61                                               Pseudomonas fluorescens 13525 56                                              Serratia marcescens 13880 54                                                  Staphylococcus aureus 12600 37                                                Staphylococcus epidermidis 14990 20                                           Streptococcus agalactiae 13813 34                                             Streptococcus faecalis 19433 20                                               Streptococcus faecium 19434 47                                                Torulopsis glabrata  2001 1.9                                                 Ureaplasma urealyticum 27618 26                                             ______________________________________                                    

Table 60 shows that probe 3 detects phylogenetically diverse bacteriaand does not hybridize to human rRNA.

                  TABLE 60                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 3 TO RNAs OF A CROSS                           SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS.                                                              % Probe*                                        Organism ATCC# Bound                                                        ______________________________________                                        Acinetobacter calcoaceticus                                                                       23055   69                                                  Bacillus subtilis  6051 35                                                    Bacteroides fragilis 23745 1.2                                                Branhamella catarrhalis 25238 43                                              Campylobacter jejuni 33560 55                                                 Chlamydia trachomatis VR878 42                                                Chromobacterium violaceum 29094 69                                            Clostridium perfringens 13124 62                                              Corynebacterium xerosis  373 23                                               Deinococcus radiodurans 35073 30                                              Derxia gummosa 15994 67                                                       Gardnerella vaginalis 14018 40                                                Hafnia alvei 13337 56                                                         Lactobacillus acidophilus  4356 36                                            Moraxella osloensis 19976 64                                                  Mycobacterium smegmatis 14468 77                                              Mycoplasma hominis 14027 1.5                                                  Neisseria gonorrhoeae 19424 26                                                Rahnella aauatilis 33071 66                                                   Rhodospirillum rubrum 11170 51                                                Vibrio parahaemolyticus 17802 68                                              Yersinia enterocolitica  9610 68                                              Human  0.9                                                                  ______________________________________                                    

Table 61 shows that probe 4 hybridizes to the RNA of bacteria commonlyfound in urine and does not detect yeast rRNA.

                  TABLE 61                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 4                                              TO RNA OF ORGANISMS FOUND IN URINE.                                                                        % Probe                                          Organism ATCC# Bound                                                        ______________________________________                                        Candida albicans   18804   4.5                                                  Candida krusei 34135 2.5                                                      Candida parapsilosis 22019 2.7                                                Candida tropicalis  750 2.5                                                   Citrobacter freundii  8090 55                                                 Enterobacter aerogenes 13048 52                                               Enterobacter cloacae 13047 57                                                 Escherichia coli 11775 70                                                     Klebsiella oxytoca 13182 70                                                   Klebsiella pneumoniae 13883 43                                                Morganella morganii 25830 74                                                  Proteus mirabilis 29906 74                                                    Proteus vulgaris 13315 73                                                     Providencia stuartii 29914 73                                                 Pseudomonas aeruginosa 10145 76                                               Pseudomonas fluorescens 13525 79                                              Serratia marcescens 13880 74                                                  Staphylococcus aureus 12600 73                                                Staphylococcus epidermidis 14990 73                                           Streptococcus agalactiae 13813 70                                             Streptococcus faecalis 19433 37                                               Streptococcus faecium 19434 63                                                Torulopsis glabrata  2001 2.2                                                 Ureaplasma urealyticum 27618 43                                             ______________________________________                                    

Table 62 shows that probe 4 detects phylogenetically diverse bacteriaand does not hybridize to human rRNA.

                  TABLE 63                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 4 TO RNAs OF A CROSS                           SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS                                                               % Probe                                         Organism ATCC# Bound                                                        ______________________________________                                        Acinetobacter calcoaceticus                                                                       23055   69                                                  Bacillus subtilis  6051 55                                                    Bacteroides fragilis 23745 3.0                                                Branhamella catarrhalis 25238 59                                              Campylobacter jejuni 33560 65                                                 Chlamydia trachomatis VR878 50                                                Chromabacterium violaceum 29094 61                                            Clostridium perfringens 13124 57                                              Corynebacterium xerosis  373 9.5                                              Deinococcus radiodurans 35073 63                                              Derxia gummosa 15994 65                                                       Gardnerella vaginalis 14018 57                                                Hafnia alvei 13337 67                                                         Lactobacillus acidophilus  4356 68                                            Moraxella osloensis 19976 68                                                  Mycobacterium smegmatis 14468 28                                              Mycoplasma hominis 14027 74                                                   Neisseria gonorrhoeae 19424 76                                                Rahnella aquatilis 33071 68                                                   Rhodospirillum rubrum 11170 59                                                Vibrio parahaemolyticus 17802 75                                              Yersinia enterocolitica  9610 74                                              Human  2.8                                                                  ______________________________________                                    

Table 63 shows that probe 5 hybridizes to the RNA of bacteria commonlyfound in urine and does not detect yeast rRNA.

                  TABLE 63                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 5                                              TO RNA OF ORGANISMS FOUND IN URINE.                                                                        % Probe                                          Organism ATCC# Bound                                                        ______________________________________                                        Candida albicans   18804   1.8                                                  Candida krusei 34135 1.7                                                      Candida parapsilosis 22019 2.2                                                Candida tropicalis  750 1.8                                                   Citrobacter freundii  8090 39                                                 Enterobacter aerogenes 13048 38                                               Enterobacter cloacae 13047 43                                                 Escherichia coli 11775 31                                                     Klebsiella oxytoca 13182 38                                                   Klebsiella pneumoniae 13883 66                                                Morganella morganii 25830 50                                                  Proteus mirabilis 29906 44                                                    Proteus vulgaris 13315 52                                                     Providencia stuartii 29914 44                                                 Pseudomonas aeruginosa 10145 47                                               Pseudomonas fluorescens 13525 25                                              Serratia marcescens 13880 35                                                  Staphylococcus aureus 12600 26                                                Staphylococcus epidermidis 14990 37                                           Streptococcus agalactiae 13813 29                                             Streptococcus faecalis 19433 14                                               Streptococcus faecium 19434 33                                                Torulopsis glabrata  2001 2.2                                                 Ureaplasma urealyticum 27618 73                                             ______________________________________                                    

Table 64 shows that probe 5 detects phylogenetically diverse bacteriaand does not hybridize to human RNA.

                  TABLE 64                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 5 TO RNAs OF A CROSS                           SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS                                                               % Probe                                         Organism ATCC# Bound                                                        ______________________________________                                        Acinetobacter calcoaceticus                                                                       23055   20                                                  Bacillus subtilis  6051 53                                                    Bacteroides fragilis 23745 44                                                 Branhamella catarrhalis 25238 22                                              Campylobacter jejuni 33560 35                                                 Chlamydia trachomatis VR878 59                                                Chromabacterium violaceum 29094 63                                            Clostridium perfringens 13124 57                                              Corynebacterium xerosis  373 1.7                                              Deinococcus radiodurans 35073 5.7                                             Derxia gummosa 15994 14                                                       Gardnerella vaginalis 14018 1.6                                               Hafnia alvei 13337 44                                                         Lactobacillus acidophilus  4356 1.5                                           Moraxella osloensis 19976 7.2                                                 Mycobacterium smegmatis 14468 39                                              Mycoplasma hominis 14027 21                                                   Neisseria gonorrhoeae 19424 40                                                Rahnella aquatilis 33071 55                                                   Rhodospirillum rubrum 11170 17                                                Vibrio parahaemolyticus 17802 66                                              Yersinia enterocolitica  9610 64                                              Human  1.6                                                                  ______________________________________                                    

Table 65 shows that probe 6 hybridizes to the RNA of bacteria commonlyfound in urine and does not detect yeast rRNA.

                  TABLE 65                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 6                                              TO RNA OF ORGANISMS FOUND IN URINE                                                                         % Probe                                          Organism ATCC# Bound                                                        ______________________________________                                        Candida albicans   18804   3.0                                                  Candida krusei 34135 2.0                                                      Candida parapsilosis 22019 2.2                                                Citrobacter freundii  8090 54                                                 Enterobacter aerogenes 13048 50                                               Enterobacter cloacae 13047 58                                                 Escherichia coli 11775 63                                                     Klebsiella oxytoca 13182 54                                                   Klebsiella pneumoniae 13883 55                                                Morganelia morganii 25830 60                                                  Proteus mirabilis 29906 64                                                    Proteus vulgaris 13315 67                                                     Providencia stuartii 29914 64                                                 Pseudomonas aeruginosa 10145 65                                               Pseudomonas fluorescens 13525 31                                              Serratia marcescens 13880 67                                                  Staphylococcus aureus 12600 53                                                Staphylococcus epidermidis 14990 34                                           Streptococcus agalactiae 13813 31                                             Streptococcus faecium 19434 18                                                Torulopsis glabrata  2001 2.5                                               ______________________________________                                    

Table 66 shows that probe 6 detects some phylogenetically diversebacteria and does not hybridize to human rRNA.

                  TABLE 66                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 5 TO RNAs OF A CROSS                           SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS.                                                             % Probe                                          Organism ATCC# Bound                                                        ______________________________________                                        Acinetobacter calcoaceticus                                                                      23055   73                                                   Bacteroides fragilis 23745 7.0                                                Branhamella catarrhalis 25238 4.0                                             Deinococcus radiodurans 35073 5.5                                             Derxia gummosa 15994 3.0                                                      Gardnerella vaginalis 14018 2.0                                               Hafnia alvei 13337 3.5                                                        Lactobacillus acidophilus  4356 17                                            Moraxella osloensis 19976 62                                                  Mycoplasma hominis 14027 44                                                   Rahnella aquatilis 33071 56                                                   Yersinia enterocolitica  9610 50                                              Human  4.0                                                                  ______________________________________                                    

Table 67 shows that probe 7 hybridizes to the RNA of bacteria commonlyfound in urine and does not detect yeast rRNA.

                  TABLE 67                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 7 TO RNA                                       OF ORGANISMS FOUND IN URINE                                                                                % Probe                                          Organism ATCC# Bound                                                        ______________________________________                                        Candida albicans   18804   2.1                                                  Candida krusei 34135 2.0                                                      Candida tropicalis  750 2.2                                                   Citrobacter freundii  8090 67                                                 Enterobacter aerogenes 13048 69                                               Enterobacter cloacae 13047 78                                                 Escherichia coli 11775 75                                                     Klebsiella oxytoca 13882 79                                                   Klebsiella pneumoniae 13883 77                                                Morganella morganii 25830 76                                                  Proteus mirabilis 29906 77                                                    Proteus vulgaris 13315 79                                                     Providencia stuartii 29914 64                                                 Pseudomonas aeruginosa 10145 76                                               Pseudomonas fluorescens 13525 78                                              Serratia marcescens 13880 66                                                  Staphylococcus aureus 12600 71                                                Staphylococcus epidermidis 14990 75                                           Streptococcus agalactiae 13813 70                                             Streptococcus faecalis 19433 58                                               Streptococcus faecium 19434 68                                                Torulopsis glabrata  2001 2.4                                                 Ureaplasma urealyticum 27618 21                                             ______________________________________                                    

Table 68 shows that probe 7 detects phylogenetically diverse bacteriaand does not hybridize to human rRNA.

                  TABLE 68                                                        ______________________________________                                        HYBRIDIZATION OF BACTERIAL PROBE 7 TO RNAs OF A CROSS                           SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS.                                                             % Probe                                          Organism ATCC# Bound                                                        ______________________________________                                        Acinetobacter calcoaceticus                                                                      23055   86                                                   Bacillus subtilis  6051 83                                                    Bacteroides fragilis 23745 69                                                 Branhamella catarrhalis 25238 74                                              Campylobacter jejuni 33560 5.3                                                Chlamydia trachomatis VR878 41                                                Chromobacterium violaceum 29094 69                                            Clostridium perfringens 13124 68                                              Corynebacterium xerosis  373 23                                               Deinococcus radiodurans 35073 70                                              Derxia gummosa 15994 69                                                       Gardnerella vaginalis 14018 68                                                Hafnia alvei 13337 77                                                         Moraxella osloensis 19976 68                                                  Mycobacterium smegmatis 14468 64                                              Mycoplasma hominis 14027 4.0                                                  Neisseria gonorrhoeae 19424 53                                                Rahnella aquatilis 33071 72                                                   Rhodospirillum rubrum 11170 73                                                Vibrio parahaemolyticus 17802 67                                              Yersinia enterocolitica  9610 66                                              Human  2.2                                                                  ______________________________________                                    

Example 20

Fungi encompass a morphologically and physiologically diverse group ofsimple eucaryotic organisms. We estimate, using published sequences ofthree fungi, Neurospora crassa, Podospora, and Saccharomyces, that therRNA of fungi are 58-60% homologous to E. coli and 84-90% homologous toone another. Some fungi grow as single cells (yeasts), others asmultinuclear filaments (molds) and still others can grow as eithersingle cells or multicellular filaments (dimorphic fungi). Although manyfungi are harmless inhabitants of their environments, others are harmfuland cause disease. The presence of any fungi in some locations isundesirable or indicative of disease (e.g., culture media,pharmaceutical products, body fluids such as blood, urine orcerebrospinal fluid, and tissue biopsies). Low levels of fungi areconsidered acceptable in other products such as drinking water and foodproducts. This has created the need for a means of detecting andquantitating fungi in a sample.

The current methods for detecting and quantifying fungi involvemicroscopic examination of samples and culture on different media.Although most yeasts can be grown from clinical samples in a matter ofdays., some filamentous fungi take up to four weeks culture time, afterwhich special staining procedures, biochemical analysis and antigentests are performed. The oligonucleotide sequences below, when used in ahybridization assay, detect the five yeasts most commonly isolated inthe clinical setting, Candida albicans, Torulopsis glabrata, Candidatropicalis, Candida parapsilosis and Candida krusei. Five other fungirepresenting the Trichosporon, Blastomyces, Cryptococcus andSaccharomyces genera are also detected. The present invention allows onestep detection of these organisms and, in relation to culture, reducesthe time to identification or elimination of these fungi as the cause ofan infection. This represents a significant improvement over prior artmethods.

The four probes which hybridize to the organisms of interest wereidentified using 3 primers complementary to conserved regions on 18S or28S rRNA. Sequence 1 was obtained using an 18S primer with the sequence5'-AGA ATT TCA CCT CTG-3'. Sequence 2 was obtained using a 28S primerwith the sequence 5'-CCT TCT CCC GAA GTT ACG G-3'. Sequences 3 and 4were obtained with a 28S primer with the sequence 5'-TTC CGA CTT CCA TGGCCA CCG TCC-3'. The following sequences were characterized and shown tohybridize to fungal rRNA. The sequences of Saccharomyces cerevisiae,Saccharomyces carlsbergensis, Escherichia coli and human rRNA were usedfor comparison with the sequences of interest.

    1.  CCC GAC CGT CCC TAT TAA TCA TTA CGA TGG                                      - 2.  CGA CTT GGC ATG AAA ACT ATT CCT TCC TGT GG                              - 3.  GCT CTT CAT TCA ATT GTC CAC GTT CAA TTA AGC AAC                          AAG G                                                                        - 4.  GCT CTG CAT TCA AAC GTC CGC GTT CAA TAA AGA AAC                          AGG G                                                                 

Sequence 1, from 18S rRNA, is 30 bases in length and has a Tm of 68° C.Sequence 2, from 23S rRNA, is 32 bases in length and has a Tm of 67° C.Sequence 3, from 23S rRNA, is 40 bases in length and has a Tm of 66° C.Sequence 4, from 235 rRNA, is 40 bases in length and has a Tm of 68-C.Sequence 1 hybridizes in the region corresponding to position 845-880 ofSaccharomyces cerevisiae 18s rRNA. Sequence 2 hybridizes in the regioncorresponding to position 1960-2000 of Saccharomyces cerevisiae 28s rRNAand sequences 3 and 4 hybridize in the region of 1225-1270 of the 28srRNA.

To demonstrate the reactivity and specificity of these probes for fungalRNA, they were used in hybridization assays. ³² P- or ¹²⁵ I-labeledoligonucleotide probes were mixed with purified RNA or RNA released fromcells by standard lysis techniques in 0.2 ml of 0.48M sodium phosphatepH 6.8, 1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM EGTA and incubated at60° C. for 2 hours. Following incubation, 5 ml of 2% hydroxyapatite,0.12 M sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was addedand the samples incubated 10 minutes at 60° C. The samples werecentrifuged and the supernatants removed. Five ml of 0.12M sodiumphosphate pH 6.8, 0.02% sodium dodecyl sulfate was added, the sampleswere mixed, centrifuged and the supernatants removed. The results areshown in Table 69. Probe 1 detects all ten fungi which were tested,probe 2 detects all six of the yeasts which were tested, probe 3 detectsfive of the six yeasts, and probe 4 detects C. krusei only. Thus probe 4could be used to detect and identify C. krusei in samples, probe 1, 2 orcombination of 3 and 4 could be used to detect the yeasts, and probe 1could be used to detect any of the ten organisms listed in Table 69.

One potential use for these probes is to identify yeasts in urinesamples or other normally sterile body fluids. The probes werehybridized to a panel of bacteria most commonly isolated from urine andshown not to react (Table 70). Table 71 shows that the probes do nothybridize to phylogenetically diverse bacteria or to human RNA.

                  TABLE 69                                                        ______________________________________                                        HYBRIDIZATION OF YEAST PROBES TO YEAST RNA                                                        % Probe Bound                                             Organism       ATCC#    #1    #2   #3   #4                                    ______________________________________                                        Blastomyces dermatitidis                                                                     C.I.     25    1.4  1.5  1.5                                     Candida albicans 18804 40 63 56 2.0                                           C. krusei 34135 73 62 2.2 70                                                  C. parapsilosis 22019 71 63 65 2.0                                            C. tropicalis  750 62 71 71 2.0                                               Cryptococcus laurentii C.I. 43 1.4 1.5 1.5                                    Cryptococcus neoformans C.I. 60 1.3 1.5 1.6                                   Torulopsis glabrata  2001 61 44 62 2.0                                        Trichosporon beigelii C.I. 57 1.3 2.1 1.5                                     Saccharomyces cerevisiae C.I. 41 67 53 1.9                                  ______________________________________                                         C.I. = Clinical isolate                                                  

                  TABLE 70                                                        ______________________________________                                        HYBRIDIZATION OF FUNGAL PROBES 1-4 TO RNA                                       OF ORGANISMS FOUND IN URINE                                                                      % Probe Bound                                            Organism        ATCC#    #1    #2    #3  #4                                   ______________________________________                                        Citrobacter freundii                                                                           8090    1.5   1.7   1.5 2.1                                    Enterobacter aerogenes 13048 2.5 1.9 2.0 2.0                                  Enterobacter cloacae 13047 2.5 1.6 2.6 2.0                                    Escherichia coli 11775 3.0 2.0 1.6 1.5                                        Klebsiella oxytoca 13182 2.5 2.2 2.5 2.0                                      Klebsiella pneumoniae 13883 2.5 2.2 2.1 2.0                                   Morganella morganii 25830 2.0 2.8 1.7 1.9                                     Proteus mirabilis 29906 2.5 1.9 2.3 2.0                                       Proteus vulgaris 13315 2.0 2.2 2.0 1.5                                        Providencia stuartii 29914 3.0 1.7 2.8 2.0                                    Pseudomonas aeruqinosa 10145 2.0 1.9 1.3 2.0                                  Pseudomonas fluorescens 13525 2.5 2.7 2.1 2.0                                 Serratia marcescens 13880 2.5 1.7 1.8 2.0                                     Staphylococcus aureus 12600 2.0 1.7 1.8 2.0                                   Staphylococcus epidermidis 14990 3.0 1.5 1.3 2.0                              Streptococcus agalactiae 13813 2.5 1.9 1.3 2.5                                Streptococcus faecalis 19433 1.7 3.3 3.5 1.9                                  Streptococcus faecium 19434 2.0 2.9 2.1 1.5                                   Ureaplasma urealyticum 21618 2.1 3.1 2.4 1.8                                ______________________________________                                    

                  TABLE 71                                                        ______________________________________                                        HYBRIDIZATION OF FUNGAL PROBES 1-4 TO RNAs OF A CROSS                           SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS                                                   % Probe Bound                                             Organism        ATCC#   #1     #2    #3   #4                                  ______________________________________                                        Acinetobacter calcoaceticus                                                                   23055   2.5    2.5   2.0  1.9                                   Bacillus subtilis  6051 2.0 2.8 2.4 2.4                                       Bacteroides fragilis 23745 2.0 2.2 2.5 2.3                                    Branhamella catarrhalis 25238 2.5 3.2 1.8 1.7                                 Campylobacter jejuni 33560 2.5 2.1 2.0 1.9                                    Chlamydia trachomatis VR878 3.1 3.1 1.8 2.7                                   Chromobacterium violaceum 29094 2.5 1.7 2.0 2.2                               Clostridium perfringens 13124 1.9 2.3 1.8 1.8                                 Corynebacterium xerosis  373 1.6 4.8 1.8 1.1                                  Deinococcus radiodurans 35073 2.0 1.6 2.1 0.8                                 Derxia gummosa 15994 3.0 1.5 1.7 1.8                                          Gardnerella vaginalis 14018 2.0 2.2 1.3 1.2                                   Hafnia alvei 13337 1.0 2.5 1.7 1.6                                            Lactobacillus acidophilus  4356 2.0 2.7 2.0 1.9                               Moraxella osloensis 19976 2.0 2.1 1.9 1.8                                     Mycobacterium smegmatis 14468 1.6 1.8 1.8 1.7                                 Mycoplasma hominis 14027 1.5 1.8 1.6 1.5                                      Neisseria gonorrhoeae 19424 2.0 2.7 1.6 1.6                                   Rahnella aquatilis 33071 2.0 2.7 2.3 2.1                                      Rhodospirillum rubrum 11170 2.0 1.8 1.6 1.5                                   Vibrio parahaemolyticus 17802 2.5 3.1 1.7 1.6                                 Yersinia enterocolitica  9610 2.0 1.8 2.3 2.2                                 Human  2.0 1.8 2.1 3.0                                                      ______________________________________                                    

Two derivatives of probe 1 also were made:

CCCGACCGTCCCTATTAATCATTACGATGGTCCTAGAAAC

CCCGACCGTCCCTATTAATCATTACGATGG

The first derivative works well at 65° C., the second at 60° C.

Example 21

Gonorrhea is one of the most commonly reported bacterial infections inthe United States, with over two million cases reported annually. Thissexually transmitted disease usually results in anterior urethritis inmales and involves the cervix in females. While severe complications andeven sterility can occur in untreated individuals, asymptomaticinfections are common, resulting in carriers who unknowingly spread thedisease.

The causative agent, Neisseria gonorrhoeae, is a gram negative, oxidasepositive diplococcus with stringent growth requirements. The method usedfor diagnosis depends on the site of infection and the patient symptoms.Gonococcal urethritis in males is diagnosed with good sensitivity andspecificity using gram stain. Culture, requiring 24-72 hours, usuallymust be performed to confirm diagnosis of gonorrhea from all females andasymptomatic males. Following the detection of the organism from growthin culture, Neisseria gonorrhoeae must be identified by further testssuch as carbohydrate degradation, coagglutination, fluorescent antibodyscreens or chromogenic enzyme substrate assays.

Neisseria gonorrhoeae is particularly difficult to detect anddistinguish using a nucleic acid probe because it is very closelyrelated to N. meningitidis. Data published in Kingsbury, D. T., J.Bacteriol. 94:870-874 (1967) shows a DNA:DNA homology for the twospecies of approximately 80-94%. Under guidelines established by the AdHoc Committee on Reconciliation of Approaches to Bacterial Systematics,Int'l J. System. Bacteriol. 37:463-464 (1987), the phylogeneticdefinition of a species generally means 70% or greater DNA:DNA homology.Despite the fact that these organisms may be considered to be the samespecies under established principles, we were able to make probescapable of distinguising them.

As expected, the rRNA homology between N. gonorrhoeae and N.meningitidis is even greater because of known conserved regions. Wenoted a 1.0% difference between the 16S and a 1.1% difference betweenthe 23S rRNA sequences of N. gonorrhoeae and N. meningitidis using oursequencing data.

Making a probe for N. gonorrhoeae was complicated by the fact that insome sites where N. meningitidis and N. gonorrhoeae differed, otherNeisseria species were similar to N. gonorrhoeae. The few mismatcheswhich exist between these two species are in the most variable regions,i.e., regions which vary not only between species, but also from strainto strain. Despite the fact that some believed the species could not bedistinguished with nucleic acid probes at all, and others believed thatrRNA was too conserved to be useful in probe diagnostics, we were ableto make probes capable of differentiating N. gonorrhoeae and N.meningitidis.

The present invention has significant advantages over each of the priorart methods; the probes are more specific and much faster than culturemethods. It also is believed that the probes are more sensitive, (i.e.,able to detect a smaller number of organisms in a clinical sample) thanprior art methods.

The primers used to identify these probe sequences had the followingsequences:

    1.  GGCCGTTACCCCACCTACTAGCTAAT                                                   - 2.   GTATTACCGCGGCTGCTGGCAC                                                 - 3.   GCTCGTTGCGGGACTTAACCCACCAT                                      

Each of the rRNA sites chosen to target had at least two mismatches toE. coli, N. meningitidis, N. cinerea, N. lactamica, N. mucosa, andKingella kingae.

Oligonucleotides complementary to sequences adjacent to the proberegions were synthesized and used in the hydridization mix accoridng toHogan et al., U.S. Pat. No. 5,030,557.

The following sequences were characterized and shown to be specific forNeisseria gonorrhoeae. The phylogenetically nearest neighbors Neisseriameningitidis, N. lactamica, N. cinerea, N. mucosa, and Kingella kingaewere used for comparison with the N. gonorrhoeae sequence.

    1.      CCG CCG CTA CCC GGT AC                                                   - 2.  TCA TCG GCC GCC GAT ATT GGC                                             - 3.  GAG CAT TCC GCA CAT GTC AAA ACC AGG TA                           

Sequence 1, complementary to 16S rRNA in the region 125-150, is 17 basesin length and has a Tm of 56° C. Sequence 2, complementary to 16S rRNAin the region 455-485, is 21 bases in length and has a Tm of 63° C.Sequence 3, complementary to 16S rRNA in the region 980-1015, is 29bases in length and has a Tm of 57° C.

The reactivity and specificity of the probes for Neisseria gonorrhoeaewas demonstrated with a hybridization assay. The three oligonucleotideprobes were iodinated and mixed with unlabeled oligonucleotides ofsequence 5'-CCC CTG CTT TCC CTC TCT AGA CGT ATG CGG TAT TAG CTG ATC TTTCG-3', 5'-GCC TTT TCT TCC CTG ACA AAA GTC CTT TAC AAC CCG-3', 5'-GGC ACGTAG TTA GCC GGT GCT TAT TCT TCA GGT AC-3', and 5'-GGT TCT TCG CGT TGCATC GAA TTA ATC CAC ATC ATC CAC CGC-3', and with purified RNA in 0.48 Msodium phosphate, ph6.8, 0.5% sodium dodecyl sulfate (SDS) and incubatedat 60° C. for one hour. Following incubation, 4 ml of 2% hydroxyapatite,0.12 M sodium phosphate pH6.8, 0.02% SDS was added and the mixture wasincubated at 60° C. for 5 minutes. The samples were centrifuged and thesupernatants were removed. Five ml of wash solution (0.12 M sodiumphosphate pH6.8, 2% SDS) was added and the samples were mixed,centrifuged, and the supernatants removed. The amount of radioactivitybound to the hydroxyapatite was determined in a gamma counter.

Table 72 shows that the probes hybridize well to N. gonorrhoeae RNA anddo not hybridize to the other species tested.

                  TABLE 72                                                        ______________________________________                                        HYBRIDIZATION OF NEISSERIA GONORRHOEAE                                          PROBES 1-3 TO NEISSERIA AND KINGELLA RNAS                                       Organisms         ATCC#   % Probe Bound                                   ______________________________________                                        Kingella kingae   23332   0.09                                                  Neisseria cinerea 14685 0.04                                                  N. gonorrhoeae 19424 48.4                                                     N. lactamica 23970 0.07                                                       N. meningitidis serogroup A 13077 0.04                                        N. meningitidis serogroup B 13090 0.04                                        N. meningitidis serogroup C 13102 0.04                                        N. mucosa 19696 0.07                                                          N. subflava 14799 0.05                                                      ______________________________________                                    

The following derivatives of Neisseria probes also have been made andused:

    GAG GAT TCC GCA CAT GTC AAA ACC AGG                                              - GAG GAT TCC GCA CAT GTC AAA ACC AGG TAA                                     - CCC GCT ACC CGG TAC GTT C                                                   - CCG CTA CCC GGT ACG TTC.                                             

Although the above examples of performance were determined using thestandard assay format previously described, the specific probes may beused under a wide variety of experimental conditions. For example,additives may be included to the reaction solutions to provide optimalreaction conditions for accelerated hybridization. Such additives mayinclude buffers, chelators, organic compounds and nucleic acidprecipitating agents such as detergents, dihydroxybenzene, sodiumdodecyl sulfate, sodium diisobutyl sulfosuccinate, sodium tetradecylsulfate, sarkosyl and the alkali metal salts and ammonium salts ofSO-2₄, PO-3₄, Cl-¹ and HCOO-¹. Such additives can be utilized by oneskilled in the art to provide optimal conditions for the hybridizationreaction to take place. These conditions for accelerated hybridizationof single stranded nucleic acid molecules into double stranded moleculesare the subject of the above-noted U.S. patent application Ser. No.627,795 filed Jul. 5, 1984, continuation filed Jun. 4, 1987 (Ser. No.not yet assigned) and Ser. No. 816,711 filed Jan. 7, 1986, which areboth entitled ACCELERATED NUCLEIC ACID REASSOCIATION METHOD.

The present invention can be carried out on nonviral organisms frompurified samples or unpurified clinical samples such as sputum, feces,tissue, blood, spinal or synovial fluids serum, urine or other bodilyfluids, or other samples such as environmental or food samples. Prior tocell breakage and hybridization, the cells can be suspended or placed insolution. In the case of the unpurified samples referred to above, thecells may remain intact and untreated in their own biologicalenvironment prior to the assay.

The probes of the present invention may be used in an assay either aloneor in combination with different probes. Several individual probes alsocan be linked together during nucleic acid synthesis. This results inone probe molecule which contains multiple probe sequences, andtherefore, multiple specificities. For example, a single nucleic acidmolecule can be synthesized which contains both the Mycobacterium aviumand the Mycobacterium intracellulare sequences described in Examples 1and 2. When hybridized with either M. avium or M. intracellulare rRNAthis probe will hybridize completely. If the two probe sequences werecombined separately in an assay only one half of the mixed individualprobes will hybridize with either M. avium or M. intracellulate rRNA.Other embodiments also may be practiced within the scope of the claims.For example, probes may be labelled using a variety of labels, asdescribed within, and may be incorporated into diagnostic kits.

We claim:
 1. A method for making a probe for use in a nucleic acidhybridization assay which comprises constructing a nucleotide polymerthat is sufficiently complementary to hybridize to a region of rRNA, orthe encoding DNA, selected to distinguish one or more non-viral targetspecies from at least one non-viral non-target species, said nucleotidepolymer comprising a target-complementary sequence obtained by:a)aligning rRNA, or the encoding DNA, sequences of said one or more targetspecies and said at least one non-target species to identify a variableregion; and b) designing said target-complementary sequence of saidnucleotide polymer by substantially maximizing the complementarity ofsaid nucleotide polymer to said variable region present in said one ormore target species while substantially minimizing the complementarityof said nucleotide polymer to said variable region present in rRNA, orthe encoding DNA, sequences of said at least one non-target species,such that a duplex formed between said nucleotide polymer and said oneor more target species has a higher T_(m) than a duplex formed betweensaid nucleotide polymer and said at least one non-target species,wherein said variable region is located in an rRNA sequence, or a DNAsequence encoding for said rRNA sequence, corresponding to a targetregion selected from the group consisting of: bases 125-150 of E. coli16S rRNA or the encoding DNA; bases 175-210 of E. coli 16S rRNA or theencoding DNA; bases 185-225 of E. coli 16S rRNA or the encoding DNA;bases 190-230 of E. coli 16S rRNA or the encoding DNA; bases 600-635 ofE. coli 16S rRNA or the encoding DNA; bases 630-675 of E. coli 16S rRNAor the encoding DNA; bases 820-860 of E. coli 16S rRNA or the encodingDNA; bases 825-860 of E. coli 16S rRNA or the encoding DNA; bases830-870 of E. coli 16S rRNA or the encoding DNA; bases 975-1020 of E.coli 16S rRNA or the encoding DNA; bases 980-1010 of E. coli 16S rRNA orthe encoding DNA; bases 980-1015 of E. coli 16S rRNA or the encodingDNA; bases 995-1030 of E. coli 16S rRNA or the encoding DNA; bases1025-1060 of E. coli 16S rRNA or the encoding DNA; bases 1255-1290 of E.coli 16S rRNA or the encoding DNA; bases 270-390 of E. coli 23S rRNA orthe encoding DNA; bases 535-560 of E. coli 23S rRNA or the encoding DNA;bases 1150-1200 of E. coli 23S rRNA or the encoding DNA; bases 1440-1600of E. coli 23S rRNA or the encoding DNA; bases 1710-1750 of E. coli 23SrRNA or the encoding DNA; and bases 2190-2330 of E. coli 23S rRNA or theencoding DNA;provided that if said target region corresponds to bases125-150 of E. coli 16S rRNA or the encoding DNA, then said targetspecies is Neisseria gonorrhoeae, if said target region corresponds tobases 175-210 of E. coli 16S rRNA or the encoding DNA, then said targetspecies is Chlamydia trachomatis, if said target region corresponds tobases 185-225 of E. coli 16S rRNA or the encoding DNA, then said targetspecies is either Mycobacterium avium, Mycobacterium intracellulare, ora Mycobacterium tuberculosis complex organism, if said target regioncorresponds to bases 190-230 of E. coli 16S rRNA or the encoding DNA,then said target species is Mycoplasma pneumoniae, if said target regioncorresponds to bases 600-635 of E. coli 16S rRNA or the encoding DNA,then said target species is Chlamydia trachomatis, if said target regioncorresponds to bases 630-675 of E. coli 16S rRNA or the encoding DNA,then said target species is a Legionella organism, if said target regioncorresponds to bases 820-860 of E. coli 16S rRNA or the encoding DNA,then said target species is Mycoplasma pneumoniae, if said target regioncorresponds to bases 825-860 of E. coli 16S rRNA or the encoding DNA,then said target species is a Group D Streptococcus organism selectedfrom the group consisting of Streptococcus faecium, Streptococcusfaecalis and Streptococcus avium, if said target region corresponds tobases 830-870 of E. coli 16S rRNA or the encoding DNA, then said targetspecies is Chlamydia trachomatis, if said target region corresponds tobases 975-1020 of E. coli 16S rRNA or the encoding DNA, then said targetspecies is a Legionella organism, if said target region corresponds tobases 980-1010 of E. coli 16S rRNA or the encoding DNA, then said targetspecies is a Campylobacter organism, if said target region correspondsto bases 980-1015 of E. coli 16S rRNA or the encoding DNA, then saidtarget species is Neisseria gonorrhoeae, if said target regioncorresponds to bases 995-1030 of E. coli 16S rRNA or the encoding DNA,then said target species is Escherichia coli, if said target regioncorresponds to bases 1025-1060 of E. coli 16S rRNA or the encoding DNA,then said target species is a Mycobacterium organism, and if said targetregion corresponds to bases 1255-1290 of E. coli 16S or the encodingDNA, then said target species is Mycoplasma pneumoniae.
 2. The method ofclaim 1 wherein the percent similarity in the rRNA sequences of said atleast one nontarget species and said one or more target species isbetween 90 and 99%.
 3. The method of claim 2 wherein said at least onenontarget species is selected based on it being known to be the speciesmost closely related phylogenetically to said one or more targetspecies.
 4. The method of claim 1 comprising the further step ofverifying that said probe does not cross hybridize to said at least onenontarget species under high stringency hybridization assay conditionsto form a detectable probe:non-target duplex, wherein under said highstringency hybridization assay conditions said probe forms a detectableprobe:target duplex with nucleic acid from said one or more targetspecies.
 5. A method for making a probe for use in a hybridization assayto distinguish two or more non-viral target species of a target group ofnon-viral organisms from one or more non-viral non-target species whichcomprises constructing an oligonucleotide that is sufficientlycomplementary to hybridize a variable region of rRNA, or the encodingDNA, present in said two or more target species, said oligonucleotidecomprising a target-complementary sequence obtained by:a) aligning rRNA,or the encoding DNA, sequences of said two or more target species withrRNA, or the encoding DNA, sequences of said one or more nontargetspecies, so as to reveal inter-species hypervariability present in saidvariable region; and b) designing said target-complementary sequence ofsaid oligonucleotide by substantially maximizing complementarity of saidoligonucleotide to said variable region present in said two or moretarget species while substantially minimizing the complementarity ofsaid oligonucleotide to said variable region present in said one or morenon-target species, such that a duplex formed between saidoligonucleotide and each of said two or more target species has a higherT_(m) than a duplex formed between said oligonucleotide and said one ormore non-target species, wherein said variable region is located in anrRNA sequence, or a DNA sequence encoding for said rRNA sequence,corresponding to a target region selected from the group consisting of:bases 185-225 of E. coli 16S rRNA or the encoding DNA; bases 630-675 ofE. coli 16S rRNA or the encoding DNA; bases 825-860 of E. coli 16S rRNAor the encoding DNA; bases 975-1020 of E. coli 16S rRNA or the encodingDNA; bases 980-1010 of E. coli 16S rRNA or the encoding DNA; bases1025-1060 of E. coli 16S rRNA or the encoding DNA; bases 270-390 of E.coli 23S rRNA or the encoding DNA; bases 535-560 of E. coli 23S rRNA orthe encoding DNA; bases 1150-1200 of E. coli 23S rRNA or the encodingDNA; bases 1440-1600 of E. coli 23S rRNA or the encoding DNA; bases1710-1750 of E. coli 23S rRNA or the encoding DNA; and bases 2190-2330of E. coli 23S rRNA or the encoding DNA;provided that if said targetregion corresponds to bases 185-225 of E. coli 16S rRNA or the encodingDNA, then said two or more target species are Mycobacterium tuberculosiscomplex organisms, if said target region corresponds to bases 630-675 ofE. coli 16S rRNA or the encoding DNA, then said two or more targetspecies are Legionella organisms, if said target region corresponds tobases 825-860 of E. coli 16S rRNA or the encoding DNA, then said two ormore target species are group D Streptococcus organisms selected fromthe group consisting of Streptococcus faecium, Streptococcus faecalisand Streptococcus avium, if said target region corresponds to bases975-1020 of E. coli 16S rRNA or the encoding DNA, then said two or moretarget species are Legionella organisms, if said target regioncorresponds to bases 980-1010 of E. coli 16S rRNA or the encoding DNA,then said two or more target species are Campylobacter organisms, and ifsaid target region corresponds to bases 1025-1060 of E. coli 16S rRNA orthe encoding DNA, then said two or more target species are Mycobacteriumorganisms.
 6. The method of claim 5 comprising the further step ofverifying that said probe does not cross hybridize to said one or morenontarget species under high stringency hybridization assay conditionsto form a detectable probe:non-target duplex, wherein under said highstringency hybridization assay conditions said probe forms a detectableprobe:target duplex with nucleic acid from each of said two or moretarget species.
 7. The method of any one of claims 1, 3 or 4, whereinsaid target region corresponds to either:bases 125-150 of E. coli 16SrRNA bases 175-210 of E. coli 16S rRNA; bases 185-225 of E. coli 16SrRNA; bases 190-230 of E. coli 16S rRNA; bases 600-635 of E. coli 16SrRNA; bases 630-675 of E. coli 16S rRNA; bases 820-860 of E. coli 16SrRNA; bases 825-860 of E. coli 16S rRNA; bases 830-870 of E. coli 16SrRNA; bases 975-1020 of E. coli 16S rRNA; bases 980-1010 of E. coli 16SrRNA; bases 980-1015 of E. coli 16S rRNA; bases 995-1030 of E. coli 16SrRNA; bases 1025-1060 of E. coli 16S rRNA; bases 1255-1290 of E. coli16S rRNA; bases 270-390 of E. coli 23S rRNA; bases 535-560 of E. coli23S rRNA; bases 1150-1200 of E. coli 23S rRNA; bases 1440-1600 of E.coli 23S rRNA; bases 1710-1750 of E. coli 23S rRNA; or bases 2190-2330of E. coli 23S rRNA.
 8. The method of claim 5 wherein said one or morenontarget species is selected based on it belonging to the group oforganisms most closely related phylogenetically to said target group oforganisms.
 9. The method of any one of claims 5, 6, or 8, wherein saidtarget region corresponds to either:bases 185-225 of E. coli 16S; bases630-675 of E. coli 16S rRNA; bases 825-860 of E. coli 16S rRNA; bases975-1020 of E. coli 16S rRNA; bases 980-1010 of E. coli 16S rRNA; bases1025-1060 of E. coli 16S rRNA; bases 270-390 of E. coli 23S rRNA; bases535-560 of E. coli 23S rRNA; bases 1150-1200 of E. coli 23S rRNA; bases1440-1600 of E. coli 23S rRNA; bases 1710-1750 of E. coli 23S rRNA; orbases 2190-2330 of E. coli 23S rRNA.
 10. The method for making anoligonucleotide probe able to distinguish between a non-viral targetspecies and a non-viral non-target species belonging to the same genus,which comprises producing said oligonucleotide to comprise a nucleotidesequence designed by:a) aligning a variable region present in saidtarget species and said non-target species, wherein said variable regionis located in an rRNA sequence, or a DNA sequence encoding for said rRNAsequence, corresponding to a target region selected from the groupconsisting of: bases 65-120 of E. coli 5S rRNA or the encoding DNA;bases 60-105 of E. coli 16S rRNA or the encoding DNA; bases 125-150 ofE. coli 16S rRNA or the encoding DNA; bases 175-210 of E. coli 16S rRNAor the encoding DNA; bases 185-225 of E. coli 16S rRNA or the encodingDNA; bases 190-230 of E. coli 16S rRNA or the encoding DNA; bases450-490 of E. coli 16S rRNA or the encoding DNA; bases 455-485 of E.coli 16S rRNA or the encoding DNA; bases 600-635 of E. coli 16S rRNA orthe encoding DNA; bases 820-860 of E. coli 16S rRNA or the encoding DNA;bases 830-870 of E. coli 16S rRNA or the encoding DNA; bases 980-1015 ofE. coli 16S rRNA or the encoding DNA; bases 995-1030 of E. coli 16S rRNAor the encoding DNA; bases 1255-1290 of E. coli 16S rRNA or the encodingDNA; bases 270-390 of E. coli 23S rRNA or the encoding DNA; bases535-560 of E. coli 23S rRNA or the encoding DNA; bases 1150-1200 of E.coli 23S rRNA or the encoding DNA; bases 1440-1600 of E. coli 23S rRNAor the encoding DNA; bases 1710-1750 of E. coli 23S rRNA or the encodingDNA; and bases 2190-2330 of E. coli 23S rRNA or the encodingDNA;provided that if said target region corresponds to bases 65-120 ofE. coli 5S rRNA or the encoding DNA, then said target species isMycoplasma pneumoniae, if said target region corresponds to bases 60-150of E. coli 16S rRNA or the encoding DNA, then said target species isChlamydia trachomatis, if said target region corresponds to bases125-150 of E. coli 16S rRNA or the encoding DNA, then said targetspecies is Neisseria gonorrhoeae; if said target region corresponds tobases 185-225 of E. coli 16S rRNA or the encoding DNA, then said targetspecies is Mycobacterium avium or Mycobaterium intracellulare, if saidtarget region corresponds to bases 190-230 of E. coli 16S rRNA or theencoding DNA, then said target species is Mycoplasma pneumoniae, if saidtarget region corresponds to bases 450-490 of E. coli 16S rRNA or theencoding DNA, then said target species is Mycoplasma pneumoniae, if saidtarget region region corresponds to bases 450-490 of E. coli 16S rRNA orthe encoding DNA, then said target species is Mycoplasma pneumoniae, ifsaid target region corresponds to bases 455-485 of E. coli 16S rRNA orthe encoding DNA, then said target species is Neisseria gonorrhoeae, ifsaid target region corresponds to bases 600-636 of E. coli 16S rRNA orthe encoding DNA, then said target species is Chlamydia trachomatis, ifsaid target region corresponds to bases 820-860 of E. coli 16S rRNA orthe encoding DNA, then said target species is Mycoplasma pneumoniae, ifsaid target region corresponds to bases 830-870 of E. coli 16S rRNA orthe encoding DNA, then said target species is Chlamydia trachomatis, ifsaid target region corresponds to bases 980-1015 of E. coli 16S rRNA orthe encoding DNA, then said target species is Neisseria gonorrhoeae, ifsaid target region corresponds to bases 995-1030 of E. coli 16S rRNA orthe encoding DNA, then said target species is Escherichia coli, and ifsaid target region corresponds to bases 1255-1290 of E. coli 16S rRNA orthe encoding DNA, then said target species is Mycoplasma pneumoniae; andb) substantially maximizing complementarity of said nucleotide sequenceto said variable region present in said target species whilesubstantially minimizing complementarity of said nucleotide sequence tosaid variable region present in said non-target species, such that aduplex formed between said oligonucleotide and said target species has ahigher T_(m) than a duplex formed between said oligonucleotide and saidnon-target species.
 11. The method of claim 10, wherein said nontargetspecies is selected based on it being the species most closely relatedphylogenetically to said target species.
 12. The method of claim 10,wherein said target region corresponds to either bases 125-150 of E.coli 16S rRNA or the encoding DNA.
 13. The method of claim 12, whereinsaid target region corresponds to bases 125-150 of E. coli 16S rRNA. 14.The method of claim 10, wherein said target region corresponds to eitherbases 1255-1290 of E. coli 16S rRNA or the encoding DNA.
 15. The methodof claim 14, wherein said target region corresponds to bases 1255-1290of E. coli 16S rRNA.
 16. The method of claim 10, wherein said targetregion corresponds to either bases 270-390 of E. coli 23S rRNA or theencoding DNA.
 17. The method of claim 16, wherein said target regioncorresponds to bases 270-390 of E. coli 23S rRNA.
 18. The method ofclaim 10, wherein said target region corresponds to either bases 535-560of E. coli 23S rRNA or the encoding DNA.
 19. The method of claim 18,wherein said target region corresponds to bases 535-560 of E. coli 23SrRNA.
 20. The method of claim 10, wherein said target region correspondsto either bases 1150-1200 of E. coli 23S rRNA or the encoding DNA. 21.The method of claim 20, wherein said target region corresponds to bases1150-1200 of E. coli 23S rRNA.
 22. The method of claim 10, wherein saidtarget region corresponds to either bases 1440-1600 of E. coli 23S rRNAor the encoding DNA.
 23. The method of claim 22, wherein said targetregion corresponds to bases 1440-1600 of E. coli 23S rRNA.
 24. Themethod of claim 10, wherein said target region corresponds to eitherbases 1710-1750 of E. coli 23S rRNA or the encoding DNA.
 25. The methodof claim 24, wherein said target region corresponds to bases 1710-1750of E. coli 23S rRNA.
 26. The method of claim 10, wherein said targetregion corresponds to either bases 2190-2330 of E. coli 23S rRNA or theencoding DNA.
 27. The method of claim 26, wherein said target regioncorresponds to bases 2190-2330 of E. coli 23S rRNA.
 28. The method ofany of claims 12, 13 or 14-27, further comprising the step of labelingsaid oligonucleotide with either an isotopic label or a non-isotopiclabel.
 29. The method of claim 10, further comprising the step oflabeling said oligonucleotide with a non-isotopic label selected fromthe group consisting of fluorescent molecules, chemiluminescentmolecules, enzymes, cofactors, enzyme substrates, and haptens.
 30. Themethod of claim 29, wherein said non-isotopic label is an acridiniumester.
 31. A method for making an oligonucleotide provbe able todistinguish two or more non-viral target species belonging to a firstgenus from one or more non-viral non-target species belonging to asecond genus, which comprises producing said oligonucleotide probe tocomprise a nucleotide sequence designed by:a) aligning a variable regionpresent in each of said two or more target species and in said one ormore non-target species, wherein said variable region is located in anrRNA sequence, or a DNA sequence encoding for said rRNA sequence,corresponding to a target region selected from the group consisting of:bases 185-225 of E. coli 16S rRNA or the encoding DNA; bases 405-428 ofE. coli 16S rRNA or the encoding DNA; bases 440-475 of E. coli 16S rRNAor the encoding DNA; bases 630-675 of E. coli 16S rRNA or the encodingDNA; bases 705-735 of E. coli 16S rRNA or the encoding DNA; bases825-860 of E. coli 16S rRNA or the encoding DNA; bases 975-1020 of E.coli 16S rRNA or the encoding DNA; bases 980-1010 of E. coli 16S rRNA orthe encoding DNA; bases 1025-1060 of E. coli 16S rRNA or the encodingDNA; bases 1125-1155 of E. coli 16S rRNA or the encoding DNA; bases270-390 of E. coli 23S rRNA or the encoding DNA; bases 350-395 of E.coli 23S rRNA or the encoding DNA; bases 365-405 of E. coli 23S rRNA orthe encoding DNA; bases 535-560 of E. coli 23S rRNA or the encoding DNA;bases 540-575 of E. coli 23S rRNA or the encoding DNA; bases 1150-1200of E. coli 23S rRNA or the encoding DNA; bases 1440-1600 of E. coli 23SrRNA or the encoding DNA; bases 1570-1610 of E. coli 23S rRNA or theencoding DNA; bases 1585-1620 of E. coli 23S rRNA or the encoding DNA;bases 1710-1750 of E. coli 23S rRNA or the encoding DNA; and bases2190-2330 of E. coli 23S rRNA or the encoding DNA;provided that if saidtarget region corresponds to bases 185-225 of E. coli 16S rRNA or theencoding DNA, then said first genus is Mycobacterium; if said targetregion corresponds to bases 405-428 of E. coli 16S rRNA or the encodingDNA, then said first genus is Campylobacter, if said target regioncorresponds to bases 440-475 of E. coli 16rRNA or the encoding DNA, thensaid first genus is Campylobacter, if said target region corersponds tobases 630-675 of E. coli 16S rRNA or the encoding DNA, then said firstgenus is Legionella, if said target region corresponds to bases 705-735of E. coli 16S rRNA or the encoding DNA, then said first genus isCampylobacter, if said target region corresponds to bases 825-860 of iE. coli 16S rRNA or the encoding DNA, then said first genus isStreptococcus, if said target region corresponds to bases 975-1020 of E.coli 16S rRNA or the encoding DNA, then said first genus is Legionella,if said target region corresponds to bases 980-1010 of E. coli 16S rRNAor the encoding DNA, then said first genus is Campylobacter, if saidtarget region corresponds to bases 1025-1060 of E. coli 16S rRNA or theencoding DNA, then said first genus Mycobacterium, if said target regioncorresponds to bases 1125-1155 of E. coli 16S rRNA or the encoding DNA,then said first genus is Salmonella, if said target region correspondsto bases 350-395 of E. coli 23S rRNA or the encoding DNA, then saidfirst genus is Legionella, if said target region corresponds to bases365-405 of E. coli 23S rRNA or the encoding DNA, then said first genusis Pseudomonas, if said target region corresponds to bases 540-575 of E.coli 23S rRNA or the encodng DNA, then said first genus isMycobacterium, if said targer region corresponds to bases 1570-1610 ofE. coli 23S rRNA or the encoding DNA, then said first genus isMycobacterium, and if said target region corresponds to bases 1585-1620of E. coli 23S rRNA or the encoding DNA, then said first genus isLegionella; and b) substantially maximizing complementarity of saidnucleotide sequence to said variable region present in each of said twoor more target species while substantially minimizing thecomplementarity of said nucleotide sequence to said variable regionpresent in said one or more non-target species, such that a probe:targetduplex formed between said oligonucleotide probe and each of said two ormore target species has a higher T_(m) than a duplex formed between saidoligonucletodide probe and said one or more non-target species; whereinthe percent similarity between the rRNA of said first genus and saidsecond genus is between 90% and 99%.
 32. The method of claim 31, whereinsaid second genus is selected based on it being the phylogeneticallymost closely related genus to said first genus.
 33. The method of claim31, wherein said target region corresponds to either bases 270-390 of E.coli 23S rRNA or the encoding DNA.
 34. The method of claim 33, whereinsaid target region corresponds to bases 270-390 of E. coli 23S rRNA. 35.The method of claim 31, wherein said target region corresponds to eitherbases 535-560 of E. coli 23S rRNA or the encoded DNA.
 36. The method ofclaim 35, wherein said target region corresponds to bases 535-560 of E.coli 23S rRNA.
 37. The method of claim 31, wherein said target regioncorresponds to either bases 1150-1200 of E. coli 23S rRNA or theencoding DNA.
 38. The method of claim 37, wherein said target regioncorresponds to bases 1150-1200 of E. coli 23S rRNA.
 39. The method ofclaim 31, wherein said target region corresponds to either bases1440-1600 of E. coli 23S rRNA or the encoding DNA.
 40. The method of anyof claim 39, wherein said target region corresponds to bases 1440-1600of E. coli 23S rRNA.
 41. The method of claim 31, wherein said targetregion corresponds to either bases 1710-1750 of E. coli 23S rRNA or theencoding DNA.
 42. The method of claim 41, wherein said target regioncorresponds to bases 1710-1750 of E. coli 23S rRNA.
 43. The method ofclaim 31, wherein said target region corresponds to either bases2190-2330 of E. coli 23S rRNA or the encoding DNA.
 44. The method ofclaim 43, wherein said target region corresponds to bases 2190-2330 ofE. coli 23S rRNA.
 45. The method of claims 33-44, further comprising thestep of labeling said oligonucleotide with either an isotopic label or anon-isotopic label.
 46. The method of claim 31, further comprising thestep of labeling said oligonucleotide with a non-isotopic label selectedfrom the group consisting the fluorescent molecules, chemiluminescentmolecules, enzymes, confactors, enzyme substrates, and haptens.
 47. Themethod of claim 46, wherein said non-isotopic label is an acridiniumester.
 48. The method of claim 2, wherein said target region correspondsto bases 125-150 of E. coli 16S rRNA or the encoding DNA.
 49. The methodof claim 2, wherein said target region corresponds to bases 125-150 ofE. coli 16S rRNA.
 50. The method of claim 2, wherein said target regioncorresponds to either bases 175-210 of E. coli 16S rRNA or the encodingDNA bases 185-225 of E. coli 16S rRNA or the encoding DNA, or bases190-230 of E. coli 16S rRNA or the encoding DNA.
 51. The method of claim2, wherein said target region corresponds to either bases 175-210 of E.coli 16S rRNA, bases 185-225 of E. coli 16S rRNA or bases 190-230 of E.coli 16S rRNA.
 52. The method of claim 2, wherein said target regioncorresponds either to bases 600-635 of E. coli 16S rRNA or the encodingDNA, or bases 630-675 of E. coli 16S rRNA or the encoding DNA.
 53. Themethod of claim 2, wherein said target region corresponds to bases600-635 of E. coli 16S rRNA, or bases 630-675 of E. coli 16S rRNA. 54.The method of claim 2, wherein said target region corresponds to eitherbases 820-860 of E. coli 16S rRNA or the encoding DNA, bases 825-860 ofE. coli 16S rRNA or the encoding DNA, or bases 830-870 of E. coli 16rRNAor the encoding DNA.
 55. The method of claim 2, wherein said targetregion corresponds to either bases 820-860 of E. coli 16S rRNA, bases825-860 of E. coli 16S rRNA, or bases 830-870 of E. coli 16S rRNA. 56.The method of claim 2, wherein said target region corresponds to eitherbases 975-1020 of E. coli 16S rRNA or the encoding DNA, bases 980-1010of E. coli 16 S rRNA or the encoding DNA, bases 980-1015 of E. coli 16SrRNA or the encoding DNA, bases 995-1030 of E. coli 16S rRNA or theencoding DNA, or bases 1025-1060 of E. coli 16S rRNA or the encodingDNA.
 57. The method of claim 2, wherein said target region correspondsto either bases 975-1020 of E. coli 16S rRNA, bases 989-1010 of E. coli16S rRNA, bases 980-1015 of E. coli 16S rRNA, bases 995-1030 of E. coli16S rRNA, or bases 1025-1060 of E. coli 16S rRNA.
 58. The method ofclaim 2, wherein said target region corresponds to bases 1255-1290 of E.coli 16S rRNA or the encoding DNA.
 59. The method of claim 2, whereinsaid target region corresponds to bases 1255-1290 of E. coli 16S rRNA.60. The method of claim 2, wherein said target region corresponds tobases 270-390 of E. coli 23S rRNA or the encoding DNA.
 61. The method ofclaim 2, wherein said target region corresponds to bases 270-390 of E.coli 23S rRNA.
 62. The method of claim 2, wherein said target regioncorresponds to bases 535-560 of E. coli 23S rRNA or the encoding DNA.63. The method of claim 2, wherein said target region corresponds tobases 535-560 of E. coli 23S rRNA.
 64. The method of claim 2, whereinsaid target region corresponds to bases 1150-1200 of E. coli 23S rRNA orthe encoding DNA.
 65. The method of claim 2, wherein said target regioncorresponds to bases 1150-1200 of E. coli 23S rRNA.
 66. The method ofclaim 2, wherein said target region corresponds to bases 1440-1600 of E.coli 23S rRNA or the encoding DNA.
 67. The method of claim 2, whereinsaid target region corresponds to bases 1440-1600 of E. coli 23S rRNA.68. The method of claim 2, wherein said target region corresponds tobases 1710-1750 of E. coli 23S rRNA or the encoding DNA.
 69. The methodof claim 2, wherein said target region corresponds to bases 1710-1750 ofE. coli 23S rRNA.
 70. The method of claim 2, wherein said target regioncorresponds to bases 2190-2330 of E. coli 23S rRNA or the encoding DNA.71. The method of claim 2, wherein said target region corresponds tobases 2190-2330 of E. coli 23S rRNA.
 72. The method of claim 5, whereinsaid target region corresponds to bases 185-225 of E. coli 16S rRNA orthe encoding DNA.
 73. The method of claim 5, wherein said target regioncorresponds to bases 185-225 of E. coli 16S rRNA.
 74. The method ofclaims 5, wherein said target region corresponds to bases 630-675 of E.coli 16S rRNA or the encoding DNA.
 75. The method of claims 5, whereinsaid target region corresponds to bases 630-675 of E. coli 16S rRNA. 76.The method of claim 5, wherein said target region corresponds to bases825-860 of E. coli 16S rRNA or the encoding DNA.
 77. The method of claim5, wherein said target region corresponds to bases 825-860 of E. coli16S rRNA.
 78. The method of claim 5, wherein said target regioncorresponds to either bases 975-1020 of E. coli 16S rRNA or the encodingDNA, bases 980-1010 of E. coli 16S rRNA or the encoding DNA, or bases1025-1060 of E. coli 16S rRNA or the encoding DNA.
 79. The method ofclaim 5, wherein said target region corresponds to either bases 975-1020of E. coli 16S rRNA, bases 980-1010 of E. coli 16S rRNA or bases1025-1060 of E. coli 16S rRNA.
 80. The method of claim 5, wherein saidtarget region corresponds to bases 270-390 of E. coli 23S rRNA or theencoding DNA.
 81. The method of claim 5, wherein said target regioncorresponds to bases 270-390 of E. coli 23S rRNA.
 82. The method ofclaim 5, wherein said target region corresponds either to bases 535-560of E. coli 23S rRNA or the encoding DNA.
 83. The method of claim 5,wherein said target region corresponds to bases 535-560 of E. coli 23SrRNA.
 84. The method of claim 5, wherein said target region correspondsto bases 1150-1200 of E. coli 23S rRNA or the encoding DNA.
 85. Themethod of claim 5, wherein said target region corresponds to bases1150-1200 of E. coli 23S rRNA.
 86. The method of claim 5, wherein saidtarget region corresponds to either bases 1440-1600 of E. coli 23S rRNAor the encoding DNA.
 87. The method of claim 5, wherein said targetregion corresponds to bases 1440-1600 of E. coli 23S rRNA.
 88. Themethod of claim 5, wherein said target region corresponds to bases1710-1750 of E. coli 23S rRNA or the encoding DNA.
 89. The method ofclaim 5, wherein said target region corresponds to bases 1710-1750 of E.coli 23S rRNA.
 90. The method of claim 5, wherein said target regioncorresponds to bases 2190-2330 of E. coli 23S rRNA or the encoding DNA.91. The method of claim 5, wherein said target region corresponds tobases 2190-2330 of E. coli 23S rRNA.
 92. The method of claim 10, whereinsaid target species is Chlamydia trachomatis and said target regioncorresponds to bases 60-105 o fE. coli 16S rRNA.
 93. The method of claim10, wherein said target species is Neisseria gonorrhoeae and said targetregion corresponds to bases 125-150 of E. coli 16S rRNA.
 94. The methodof claim 10, wherein said target species wherein said target species iseither Mycobacterium avium, Mycobacterium intracellulare, Mycoplasmapneumoniae, or Chlamydia trachomatis,provided that if said targetspecies is Mycobacterium avium or Mycobacterium intracellulare, thensaid target region corresponds to bases 185-225 of E. coli 16S rRNA. ifsaid target species is Mycoplasma pneumoniae, then said target regioncorresponds to bases 190-230 of E. coli 16 rRNA, and if said targetspecies is Chlamydia trachomatis, then said target region corresponds tobases 175-210 of E. coli 16S rRNA.
 95. The method of claim 10, whereinsaid target species is either Mycoplasma pneumoniae or Neisseriagonorrhoeae,provided that if said target species is Mycoplasmapneumoniae, then said target region corresponds to bases 450-490 of E.coli 16S rRNA, and if said target species is Neisseria gonorrhoeae, thensaid target region corresponds to bases 455-485 of E. coli 16S rRNA. 96.The method of claim 10, wherein said target species is Chlamydiatrachomatis and said target region corresponds to bases 600-635 of E.coli 16S rRNA.
 97. The method of claim 10, wherein said target speciesis either Mycoplasma pneumoniae or Chlamydia trachomatis,provided thatif said target species is Mycoplasma pneumoniae, then said target regioncorresponds to bases 820-860 of E. coli 16S rRNA, and if said targetspecies is Chlamydia trachomatis, then said target region corresponds tobases 830-870 of E. coli 16S rRNA.
 98. The method of claim 10, whereinsaid target species is either Escherichia coli or Neisseriagonorrhoeae,provided that if said target species is Escherichia coli,then said target region corresponds to bases 995-1030 of E. coli 16SrRNA, and if said target species is Neisseria gonorrhoeae, then saidtarget region corresponds to bases 980-1015 of E. coli 16S rRNA.
 99. Themethod of claim 10, wherein said target species is either Proteusmirabilis, Chlamydia trachomatis or Enterobacter cloacae,provided thatif said target species is Proteus mirabilis, then said target regioncorresponds to bases 270-305 of E. coli 23S rRNA, if said target speciesis Chlamydia trachomatis, then said target region corresponds to eitherbases 275-320, or 330-365, of E. coli 23S rRNA, and if said targetspecies is Enterobacter cloacae, then said target region corresponds tobases 305-340 of E. coli 23S rRNA.
 100. The method of claim 10, whereinsaid target species is Chlamydia trachomatis and said target regioncorresponds to bases 1160-1190 of E. coli 23S rRNA.
 101. The method ofclaim 10, wherein said target species is Chlamydia trachomatis and saidtarget region corresponds to either bases 1450-1490, or 1510-1545, of E.coli 23S rRNA.
 102. The method of claimss 24 o 25, wherein said targetspecies is Chlamydia trachomatis.
 103. The method of claim 31, whereinsaid first genus is Mycobacterium and said target region corresponds tobases 185-225 of E. coli 16S rRNA.
 104. The method of claim 31, whereinsaid first genus is Campylobacter and said target region corresponds toeither bases 405-428, or 440-475, of E. coli 16S rRNA.
 105. The methodof claim 31, wherein said first genus is Legionella and said targetregion corresponds to bases 630-675 of E. coli 16S rRNA.
 106. The methodof claim 31, wherein said first genus is Streptococcus and said targetregion corresponds to bases 825-860 of E. coli 16S rRNA.
 107. The methodof claim 31, wherein said first genus is either Legionella,Campylobacter or Mycobacterium,provided that if said target species isLegionella, then said target region corresponds to bases 975-1020 of E.coli 16S rRNA, if said target species is Campylobacter, then said targetregion corresponds to bases 980-1010, of E. coli 16S rRNA, and if saidtarget species is Mycobacterium, then said target region corresponds tobases 1025-1060 of E. coli 16S rRNA.
 108. The method of claim 31,wherein said first genus is either Legionella, Salmonella, orPseudomonas,provided that if said first genus is Legionella, then saidtarget region corresponds to bases 350-395 of E. coli 23S rRNA, if saidfirst genus is Salmonella, then said target region corresponds to bases335-375 of E. coli 23S rRNA, and if said first genus is Pseudomonas,then said target region corresponds to bases 365-405 of E. coli 23SrRNA.
 109. The method of claim 31, wherein said first genus isMycobacterium and said target region corresponds to bases 540-575 of E.coli 23S rRNA.
 110. The method of claim 31, wherein said first genus isMycobacterium and said target region corresponds to bases 1155-1190 ofE. coli 23S rRNA.
 111. The method of claim 31, wherein said first genusis either Legionella or Mycobacterium,provided that if said first genusis Mycobacterium, then said target region corresponds to either bases1440-1475, 1515-1555, or 1570-1610, of E. coli 23S rRNA, and if saidfirst genus is Legionella, then said target region corresponds to bases1585-1620 of E. coli 23S rRNA.
 112. The method of claim 31, wherein saidfirst genus is either Legionella or Mycobacterium,provided that if saidfirst genus is Mycobacterium, then said target region corresponds tobases 2195-2235 of E. coli 23S rRNA, and if said first genus isLegionella, then said target region corresponds to bases 2280-2330 of E.coli 23S rRNA.
 113. The method of claim 10, wherein said target speciesis Chlamydia trachomatis and said target region corresponds to bases60-105 of E. coli 16S rRNA or the encoding DNA.
 114. The method of claim10, wherein said target species is Neisseria gonorrhoeae and said targetregions corresponds to bases 125-150 of E. coli 16S rRNA or the encodingDNA.
 115. The method of claim 5, wherein said target region correspondsto bases 270-390 of E. coli 23S rRNA.
 116. The method of claim 10,wherein said target species is either Mycoplasma pneumoniae or Neisseriagonorrhoeae,provided that if said target species is Mycoplasmapneumoniae, then said target region corresponds to bases 450-490 of E.coli 16S rRNA or the encoding DNA, and if said target species isNeisseria gonorrhoeae, then said target region corresponds to bases455-485 of E. coli 16S rRNA or the encoding DNA.
 117. The method ofclaim 10, wherein said target species is Chlamydia trachomatis and saidtarget region corresponds to bases 600-635 of E. coli 16S rRNA or theencoding DNA.
 118. The method of claim 10, wherein said target speciesis either Mycoplasma pneumoniae or Chlamydia trachomatis,provided thatif said target species is Mycoplasma pneumoniae, then said target regioncorresponds to bases 820-860 of E. coli 16S rRNA or the encoding DNA,and if said target species is Chlamydia trachomatis, then said targetregion corresponds to bases 830-870 of E. coli 16S rRNA or the encodingDNA.
 119. The method of claim 10, wherein said target species is eitherEscherichia coli or Neisseria gonorrhoeae,provided that if said targetspecies is Escherichia coli, then said target region corresponds tobases 995-1030 of E. coli 16S rRNA or the encoding DNA, and if saidtarget species is Neisseria gonorrhoeae, then said target regioncorresponds to bases 980-1015 of E. coli 16S rRNA or the encoding DNA.120. The method of claim 10, wherein said target species is eitherProteus mirabilis, Chlamydia trachomatis or Enterobactercloacae,provided that if said target species is Proteus mirabilis, thensaid target region corresponds to bases 270-305 of E. coli 23S rRNA orthe encoding DNA, if said target species is Chlamydia trachomatis, thensaid target region corresponds to either bases 275-320, or 330-365, ofE. coli 23S rRNA or the encoding DNA, and if said target species isEnterobacter cloacae, then said target region corresponds to bases305-340 of E. coli 23S rRNA or the encoding DNA.
 121. The method ofclaim 10, wherein said target species is Chlamydia trachomatis and saidtarget region corresponds to bases 1160-1190 of E. coli 23S rRNA or theencoding DNA.
 122. The method of claim 10, wherein said target speciesis Chlamydia trachomatis and said target region corresponds to eitherbases 1450-1490, or 1510-1545, of E. coli 23S rRNA or the encoding DNA.123. The method of claim 31, wherein said first genus is Mycobacteriumand said target region corresponds to bases 185-225 of E. coli 16S rRNAor the encoding DNA.
 124. The method of claim 31, wherein said firstgenus is Campylobacter and said target region corresponds to eitherbases 405-428, or 440-475, of E. coli 16S rRNA or the encoding DNA. 125.The method of claim 31, wherein said first genus is Legionella and saidtarget region corresponds to bases 630-675 of E. coli 16S rRNA or theencoding DNA.
 126. The method of claim 31, wherein said first genus isStreptococcus and said target region corresponds to bases 825-860 of E.coli 16S rRNA or the encoding DNA.
 127. The method of claim 31, whereinsaid first genus is either Legionella, Campylobacter orMycobacterium,provided that if said target species is Legionella, thensaid target region corresponds to bases 975-1020 of E. coli 16S rRNA orthe encoding DNA, if said target species is Campylobacter, then saidtarget region corresponds to bases 980-1010 of E. coli 16S rRNA or theencoding DNA, and if said target species is Mycobacterium, then saidtarget region corresponds to bases 1025-1060 of E. coli 16S rRNA or theencoding DNA.
 128. The method of claim 31, wherein said first genus iseither Legionella, Salmonella, or Pseudomonas,provided that if saidfirst genus is Legionella, then said target region corresponds to bases350-395 of E. coli 23S rRNA or the encoding DNA, if said first genus isSalmonella, then said target region corresponds to bases 335-375, of E.coli 23S rRNA or the encoding DNA, and if said first genus isPseudomonas, then said target region corresponds to bases 365-405 of E.coli 23S rRNA or the encoding DNA.
 129. The method of claim 31, whereinsaid first genus is Mycobacterium and said target region corresponds tobases 540-575 of E. coli 23S rRNA or the encoding DNA.
 130. The methodof claim 31, wherein said first genus is Mycobacterium and said targetregion corresponds to bases 1155-1190 of E. coli 23S rRNA or theencoding DNA.
 131. The method of claim 31, wherein said first genus iseither Legionella or Mycobacterium,provided that if said first genus isMycobacterium, then said target region corresponds to either bases1440-1475, 1515-1555, or 1570-1610, of E. coli 23S rRNA or the encodingDNA, and if said first genus is Legionella, then said target regioncorresponds to bases 1585-1620 of E. coli 23S rRNA or the encoding DNA.132. The method of claim 31, wherein said first genus is eitherLegionella or Mycobacterium,provided that if said first genus isMycobacterium, then said target region corresponds to bases 2195-2235 ofE. coli 23S rRNA or the encoding DNA, and if said first genus isLegionella, then said target region corresponds to bases 2280-2330 of E.coli 23S rRNA or the encoding DNA.
 133. The method of claim 10, whereinsaid target region corresponds bases 65-120 of E. coli 5S rRNA or theencoding DNA.
 134. The method of claim 133, wherein said target regioncorresponds bases 65-120 of E. coli 5S rRNA.
 135. The method of claim31, wherein said first genus is Campylobacter and said target regioncorresponds to bases 705-735 of E. coli 16S rRNA or the encoding DNA.136. The method of claim 135, wherein said target region corresponds tobases 705-735 of E. coli 16S rRNA.
 137. The method of claim 31, whereinsaid first genus is Salmonella and said target region corresponds tobases 1125-1155 of E. coli 16S rRNA or the encoding DNA.
 138. The methodof claim 137, wherein said target region corresponds to bases 1125-1155of E. coli 16S rRNA.
 139. The method of claim 10, wherein said targetregion corresponds to either bases 125-150 of E. coli 16S rRNA or theencoding DNA.
 140. The method of claim 139, wherein said target regioncorresponds to bases 125-150 of E. coli 16S rRNA.